Background: Preschools and childcare settings offer opportunities to promote adequate levels of physical activity. Research is needed to identify the key features of these settings to optimize young children's activity. The aims of this study were to determine if differences existed in preschool children's physical activity during care hours compared with outside care hours and to examine a comprehensive range of potential center-based correlates of physical activity for preschool boys and girls. Methods: Data are from the Healthy Active Preschool and Primary Years study: 71 childcare centers, 65 preschools and 1002 preschool children. Percent of time in total (light- to vigorous-intensity) physical activity was measured using Actigraph GT1M accelerometers. Center physical environment characteristics, policies and practices were assessed by trained research staff using comprehensive audit tools. Data were collected in 2008/9 and were analyzed separately for boys and girls in Stata using multilevel mixed effects models. Results: Boys and girls were less active during care than outside care hours (51.1 % vs. 52.4 %, p = 0.01; 48.0 % vs. 51.5 %, p < 0.0001, respectively). In the final adjusted models, number of outdoor spaces with natural ground coverings was associated with boys' physical activity (coeff = 0.477, 95 % CI 0.089, 0.867) and the amount of time girls spent indoors before going outdoors was inversely associated with their physical activity (coeff = -0.035, 95 % CI -0.065, -0.004). The models explained 12 and 10 % of boys' and girls' physical activity during care hours, respectively. Conclusions: This study identified that children are significantly less active during than outside care hours. Few center-based correlates of preschool children's physical activity were identified. Future research should explore other aspects of centers, such as what children actually do while they are outside, and broader potential influences on children's behaviours including social, cultural and policy contexts within which centers operate. (author abstract)