Millions of American children spend much of their day in structured child care centers; therefore, an understanding of the physical activity (PA) levels of children attending such centers takes on a high level of public health significance. PURPOSE: To describe the PA levels of children during preschool attendance and to compare their PA levels between school days and comparable hours on a weekend day. METHODS: Participants were 298 children, 3- to 5-years of age (50% male; 49% African American) from 20 preschools. Children wore accelerometers at preschool and at home over a 2-week period. Arrival and departure times from the preschool were recorded for each child using parent's sign in and out sheets. In-school minutes per hour of sedentary, light, and moderate-to-vigorous physical activity (MVPA) were calculated. A subset of children (n = 223) provided data for both preschool days and weekend days, and the time period from 10:00 AM to 5:00 PM was used for analyses. Mixed model ANOVA was used to analyze group differences, and repeated measure mixed model ANOVA was used to compare PA variables on preschool days and weekend days. Preschool was a random variable in all mixed models. RESULTS: Children wore accelerometers for an average of 5.5 days and approximately 8.1 hours per day. While in preschool, children engaged in 7.4 ± 2.5 min·hr-1 of MVPA, 19.3 ± 4.1 min·hr-1 of light PA, and 33.3 ± 5.8 min·hr-1 of sedentary behavior. During preschool attendance, boys spent more time in MVPA (7.9 ± 0.3 min·hr-1) and less time in sedentary behavior (32.9 ± 0.8 min·hr-1) than girls (6.9 ± 0.3 min·hr-1 and 34.1 ± 0.8 min·hr-1), respectively (p <.05). There were no gender differences for light PA. Levels of PA did not differ between preschool days and weekend days for MVPA (6.3 ± 0.3 min·hr-1 vs. 6.9 ± 0.3 min·hr-1), light PA (17.0 ± 0.6 min·hr-1 vs. 18.0 ± 0.6 min·hr-1), and sedentary behavior (34.6 ± 0.8 min·hr-1 vs. 33.1 ± 0.8 min·hr-1). CONCLUSIONS: During the preschool day, children spent less than 10 minutes per hour in MVPA and more than 30 minutes per hour in sedentary behavior. Approximately one-third of each hour during the preschool day was spent in light PA. Activity levels were very similar on school and weekend days.