The purpose of this study was to explore the influences of caregiver-child interaction and temperament on cortisol concentrations of toddlers in full-day childcare. Sensitive and responsive caregiving is critical to the quality of children's early experiences. Caregivers who are attuned to unique needs of young children can support and guide child growth and development. In the context of relationships the child grows, develops, and learns to biologically respond to events in his or her world that he or she may perceive as threatening, with the production of cortisol. Studies show that regulation of cortisol release later in life may be shaped by social experiences during early development. This exploratory study investigated the influences of caregiver-child interaction and temperament as measured by the Early Childhood Behavior Questionnaire (ECBQ) on cortisol concentrations in toddlers in full-day childcare. Seventy-three (31female, 42 male) toddlers, in 11 full-day childcare classrooms in communities in southern Appalachia participated. Classrooms were evaluated using the toddler Classroom Assessment Scoring System (CLASS) and an adapted version of the Engagement Check II. Morning and afternoon cortisol concentration was measured in enzymeimmunoassays of saliva. Findings indicated that as years of teacher experience increased, cortisol concentrations decreased. Likewise, as the percentage of time toddlers were engaged in developmentally appropriate activities increased, cortisol concentration decreased. Mean cortisol concentrations decreased from mid-morning to mid-afternoon. Findings of individual cortisol concentrations were mixed. Temperament data showed a relationship between management of arousal or impulse control (surgency) and cortisol concentration. No statistically significant correlation was found between cortisol concentration and the dimensions of the toddler CLASS. Regression analysis of the 3 dimensions of temperament (negative affect, effortful control, surgency) showed surgency to be related to cortisol concentration. The study adds to the body of research on very young children in full-day childcare and elevated cortisol concentrations by including children in communities in southern Appalachia as well as measuring teacher-child interaction in childcare using the newly released toddler CLASS. Future research is needed to delineate the developmental outcomes and long-term impact of excess stress in this population.