The effects of long-term endurance exercise training, body composition, and cardiovascular fitness (Vo₂max) on the activity of adipose tissue lipoprotein lipase (AT-LPL) and lipoprotein lipids were examined in 66 healthy age-matched middle-aged and older men (mean ± SE, 61 ± 1 years). We compared subcutaneous abdominal (ABD) and gluteal (GLT) heparin-elutable AT-LPL activity in 19 master athletes (Vo₂max > 40 mL/kg/min) and 20 lean sedentary men (Vo₂max < 40 mL/kg/min) versus 27 obese sedentary men (Vo₂max < 40 mL/kg/min; body fat > 27%). Fasting insulin and leptin levels were similar in master athletes and lean sedentary men, but were lower than in obese sedentary men. There were no differences in fasting values for total cholesterol or low-density lipoprotein cholesterol (LDL-C) among the groups, but master athletes had lower triglyceride (TG) values (P < .05) and higher high-density lipoprotein cholesterol (HDL-C) and HDL₂-C (P < .05) than obese and lean sedentary men. There were no regional (ABD v GLT) differences in the activity of AT-LPL in these groups, but obese sedentary men had higher levels of ABD AT-LPL (2.1 ± 0.3 nmol/10⁶ cells · min) than lean sedentary men (0.8 ± 0.2) and master athletes (0.5 ± 0.1, P = .01). Similar results were observed for GLT AT-LPL. Both ABD and GLT AT-LPL activity correlated positively with percent body fat (r = .46 to .54, P < .001), fasting insulin (r = .37 to .45, P < .001), and leptin (r = .61 to .65, P < .0001), but not with Vo₂max. In stepwise multiple regression analysis, leptin was the main independent predictor of ABD (R² = .43, P < .0001) and GLT (R² = .40, P < .0001) AT-LPL activity. Plasma TG correlated positively (r = .32, P < .01) and HDL-C correlated negatively (r = −.32, P = .02) with ABD AT-LPL activity, but these relationships were not significant after controlling for percent body fat or leptin. The results of this study indicate that in healthy middle-aged and older men, the major determinants of AT-LPL activity are obesity and its major associated hormones, leptin and insulin, not cardiovascular fitness, and also suggest that the higher HDL-C levels observed in endurance-trained men are not associated with increased AT-LPL activity.