The increasing prevalence of obesity has focused attention on the contribution of physical activity and its interaction with predisposing genetic factors. To examine 1) the relation between physical activity and total-body and central abdominal fat, independent of genetic and other environmental factors, and 2) the influence of physical activity in persons who are genetically susceptible to generalized or central adiposity. Cross-sectional study. A London academic teaching hospital. 970 healthy female twins (mean age, 55.5 years [range, 39 to 70 years]; body mass index, 24.4 kg/m2 [range, 16.4 to 44.0 kg/mg2]). There were 241 monozygotic pairs, 228 dizygotic pairs, and 32 women whose co-twin lacked complete data. Fifty-six percent of participants were of normal weight, 30% were overweight, 7% were obese, and 7% were underweight. Total-body and central abdominal fat were measured by dual-energy x-ray absorptiometry. Physical activity was assessed by quantitative and semiquantitative questionnaires. Data on dietary intake, socioeconomic status, smoking status, and use of hormone replacement therapy (HRT) were also gathered. Total-body and abdominal central adiposity were lower with higher levels of home, sporting, and sweating-associated activity. Total-body and central abdominal fat were 5.6 kg and 0.44 kg lower, respectively, in participants who reported vigorous weight-bearing activity. Physical activity was the strongest independent predictor of total-body fat (beta = -0.6 [CI, -1.06 to -0.15]; P = 0.009) and central abdominal fat (beta = -0.07 [CI, -0.1 to -0.03]; P < 0.001) in a regression model that included age, diet, smoking, HRT use, and socioeconomic status. Monozygotic twin pairs who were concordant for smoking and HRT status but were discordant for moderate-intensity sport showed greater within-pair differences in total-body fat than those who were concordant for activity level. In this model, 1 and 2 hours of moderate-intensity sport accounted for within-pair differences of 1.0 kg (P = 0.050) and 1.4 kg (P = 0.040), respectively, of total-body fat. In participants who had an overweight twin, higher levels of physical activity were still associated with 3.96-kg lower total-body fat and 0.53-kg lower central abdominal fat. Current physical activity predicts lower total-body and central abdominal adiposity in healthy middle-aged women. After controlling for genetic and environmental factors, the influence of physical activity was greater than that of other measured environmental factors. Participants with a genetic predisposition to adiposity did not show a lesser effect of physical activity on body fat mass.