Leptin is an adipose tissue-derived hormone that appears to regulate energy balance in rodents. The physiological role of leptin in human obesity is unclear. In order to determine the relationship between systemic leptin levels and selected biochemical and physiological variables, 305 adult Caucasian males were studied. Serum leptin was closely related to fat mass (r=0.73, P<0.05), percent fat (r=0.69, P<0.05), waist circumference (r=0.66, P<0.05), and fasting insulin (r=0.45), and was inversely related to maximal treadmill time (r=-0.46). In contrast, much weaker relationships were observed between leptin and age (r=0.11, P<0.05), total cholesterol (r=0.15, P<0.05), HDL-C (r=-0.22, P<0.05), triglyceride (r=0.12, P<0.05), and glucose (r=0.12, P<0.05). In a step-wise multiple regression analysis, fat mass was the best predictor of systemic leptin levels, and this relationship remained significant even after adjusting for treadmill time, fasting insulin, or percent fat. When subjects were divided into discrete subsets based on body fat, and subsequently classified into high and low fitness categories based on treadmill time, leptin levels did not differ between high and low fitness groups until body fat exceeded 25%. Thus, at higher levels of body fat, higher treadmill time is associated with lower systemic leptin levels, even at a similar body fat content. These data support the hypothesis that fat mass is the primary determinant of systemic leptin in adult males. Cardiorespiratory fitness levels appear to influence this relationship only at higher levels of body fat.