Mycoplasma gallisepticum (MG) is the organism that causes avian respiratory mycoplasmosis, leading to chronic respiratory disease in chickens and infectious sinusitis in turkeys (Stipkovits and Kempf, 1996; Levisohn and Kleven, 2000). It is also responsible for reductions in egg production and other economic losses in the poultry industry (Stipkovits and Kempf, 1996; Levisohn and Kleven, 2000). Commercially, layer chickens are vaccinated against MG in the pullet phase before lay. In this dissertation, the potential application of in ovo vaccination in layer embryos for the subsequent early protection of pullets against field-strain MG infections was investigated. The use of different sites of injection [air cell (AC) or amnion (AM)] and various dosages of live attenuated 6/85 (6/85MG) and ts-11 (ts-11MG) vaccine strains of MG delivered in ovo at 18 days of incubation were evaluated. Vaccine dosages of ts-11MG up to 7.25 x 105 CFU did not have negative effects on the hatch and posthatch results. However, only the 7.25 x 105 CFU dosage had a negative effect on overall BWG. The ts-11MG was not transmitted from vaccinated to sentinel birds at both the hatch and posthatch periods, and no subsequent MG DNA or serology responses were detected in response to the vaccine. Administration of the 6/85MG vaccine at dosages up to 1.73 x 103 CFU did not negatively affect hatchability or other posthatch variables. Antibody production against 6/85MG through d 42 posthatch with no associated hatch or posthatch mortalities were likewise observed. The high dose of 6/85MG (1.73 x 104 CFU) resulted in a greater than 15 % mortality at hatch and a greater than 40 % mortality during the first 2 wk posthatch. Trachea and bronchi lesion scores in the pullets were significantly increased when they were challenged at d 28 of age with RMG. However, birds that were in ovo-vaccinated with 6/85MGV exhibited no significant microscopic lesions due to the RMG challenge. The 1.66 x 103 CFU dosage of 6/85MGV is proposed to offer the best protection in layer pullets against field-strain MG infections.