The contribution of an organized after-school physical activity program to total daily energy expenditure (TDEE) was estimated from a sample of 27, 12-to-13-year-old females. TDEE was estimated using a 1-day activity recall log and daily step counts were measured using the Yamax Pedometer. Data were collected across three consecutive school days. Given the small sample size, a measure of practical significance, effect size (ES), was computed using point-biserial (rpb) correlation coefficient. A significant difference was found between participants reported Met Minutes (MM) on the day the after-school program was held (M=2.06, SD=.39), and non-program days (M=1.82, SD=.25, p=.005, rpb=.38). A significant difference was also found between steps accrued on the day the after-school program was held (M=11, 599; SD=2,584) and days the program was not held (M=9,848; SD=1976, p=.04, rpb=.22). Girls who participated in the after-school program had greater TDEE and accrued more total daily steps than on the days that they did not participate in the after-school program. Therefore, given the potential impact of sex-segregated environments on physical activity participation in adolescent girls, and the dearth of information regarding the efficacy of such programs, results from this study suggest that organized, after-school programs may contribute significantly toward overall physical activity levels and daily energy expenditure of adolescent females.