Declining levels of physical activity probably contribute to the increasing prevalence of overweight in US youth. In this study, the authors examined cross-sectional and longitudinal associations between physical activity and body composition in sixth- and eighth-grade girls. In 2003, girls were recruited from six US states as part of the Trial of Activity for Adolescent Girls. Physical activity was measured using 6 days of accelerometry, and percentage of body fat was calculated using an age- and ethnicity-specific prediction equation. Sixth-grade girls with an average of 12.8 minutes of moderate-to-vigorous physical activity (MVPA) per day (15th percentile) were 2.3 times (95% confidence interval: 1.52, 3.44) more likely to be overweight than girls with 34.7 minutes of MVPA per day (85th percentile), and their percent body fat was 2.64 percentage points greater (95% confidence interval: 1.79, 3.50). Longitudinal analyses showed that percent body fat increased 0.28 percentage points less in girls with a 6.2-minute increase in MVPA than in girls with 4.5-minute decrease (85th and 15th percentiles of change). Associations between MVPA in sixth grade and incidence of overweight in eighth grade were not detected. More population-based research using objective physical activity and body composition measurements is needed to make evidence-based physical activity recommendations for US youth