<p dir="ltr">Due to increasing legislative and societal demand for humane egg production, an increased production of cage-free eggs is inevitable. Past research has focused on the impact of cage-free housing systems (CFS) on laying hens' performance, health, and space use; however, limited studies have examined the impacts of CFS on the behavioral activities of laying hens, particularly in single-tier floor systems in the U.S. Broadly, this research examined the influence of two housing systems on the behavior of laying hens as they age. Specifically, the study aimed to provide information on how dissimilar cage-free systems meet the behavioral needs of laying hens. </p><p dir="ltr">Two hundred pullets were randomly assigned to either a single-tier floor room (SFR) or a modified multi-tier aviary room (MAR) at 17 weeks of age (WOA), where they remained until 85 WOA. Starting at 55 WOA, thirty focal birds were marked with non-toxic markers for individual identification in each room. The behavior of the birds was recorded continuously over two consecutive days during the mid-laying (55 WOA) and late-laying (85 WOA) phases. The behavioral expressions of the focal birds were annotated using 10-min continuous sampling at 5 time points (10 minutes after lights-on (TOD 1), 4 hr after lights-on (TOD 2), 8hr after lights-on (TOD 3), 12 hr after lights-on (TOD 4), and 10 minutes before lights-off (TOD 5)). At each of these 5 time points, 4 birds were randomly selected for observation. Behavior duration and frequency data were analyzed using PROC GLIMMIX in SAS 9.4, with the main effects of housing system (HS), age, and time of day (TOD). Descriptive statistics were used to present the behavioral time budgets of marked and unmarked birds in MAR and SFR. Further, the behaviors performed in different spatial zones (areas within each room) were presented as the percentage of observed time laying hens spent engaged in a specific behavior over the total time at a particular spatial zone. </p><p dir="ltr">Birds in MAR and SFR spent most of the observed time perching at all ages. The birds (marked and unmarked) performed environmental pecking and foraging for the highest percentage of time in the litter area of SFR and MAR. Similarly, the percentage of time the birds (marked and unmarked) spent preening and performing OCB (wing flapping, stretching, and feather-ruffling) was highest when they occupied the litter zone of MAR and SFR. Housing system influenced the time that both marked and unmarked birds spent performing foraging and environmental pecking (EP) such that marked and unmarked birds in SFR spent more time engaged in these behaviors than marked and unmarked birds in MAR (Marked: SFR vs. MAR, P = 0.043; Unmarked: SFR vs. MAR, P = 0.002). Further, marked and unmarked birds in SFR exhibited a higher frequency of EP than birds in MAR (Marked: SFR vs. MAR, P = 0.02; Unmarked: SFR vs. MAR, P < 0.001). The frequency of locomotion (standing and walking) activity differed between SFR and MAR birds. Birds (marked and unmarked) stood more frequently in SFR than in MAR (Marked: SFR vs. MAR, P = 0.046; Unmarked: SFR vs. MAR, P < 0.001). Further, SFR birds had a higher walking frequency than MAR birds (Marked: P = 0.003; Unmarked: P = 0.02). Birds (marked and unmarked) in MAR perched more frequently than SFR birds (Marked: SFR vs. MAR, P < 0.001; Unmarked: SFR vs. MAR, P = 0.01); however, unmarked birds preened more frequently in SFR than in MAR (P < 0.001). Regarding time of day, both marked and unmarked birds exhibited higher preening frequency 10 mins after lights-on (TOD 1) than at other times of day (P < 0.001). Similarly, marked birds perched for a longer duration early in the morning (TOD 1) and late at night (TOD 5), than at TOD 2 and TOD 3 (P < 0.001). </p><p dir="ltr">This study revealed that MAR and SFR may differ in how they accommodate the behavioral needs of laying hens, with hen activity levels varying between the two types of housing systems. Further research is needed to understand the implications of behavioral outcomes for the welfare of laying hens in different types of CFS.</p>