Abstract We examined possible determinants of serum high density lipoprotein cholesterol (HDL-C) concentrations in 56 male distance runners (aged 20–56 years) by comparing runners whose HDL-C were either above or below the group median of 63 ± 13 (± SD) mg/dl. HDL-C averaged 53 ± 7 mg/dl for runners below and 73 ± 11 mg/dl for runners above the median. Neither exercise training (miles run per week, years of running), physical characteristics (height, weight, adiposity), or dietary factors (total daily caloric intake and daily caloric intake from protein, fat, saturated fat, polyunsaturated fat, carbohydrate, and alcohol) differed between the two groups ( P>0.05, MANOVA). Apo A-1 ( P < 0.01) was higher and triglyceride concentrations lower ( P = 0.07) in the high HDL-C group. The data were also analyzed by comparing runners in the lowest and highest tertiles for HDL-C values and essentially the same results were obtained. When all runners were combined, neither training, physical characteristics nor dietary intake was significantly related to HDL-C ( P>0.05). Total cholesterol and apo A-1 were directly related ( r = 0.35 and r = 0.66, respectively, P < 0.01) and triglycerides inversely related ( r = −0.31, P < 0.05) to HDL-C. Plasma post-heparin lipoprotein lipase activity (LPLA), hepatic triglyceride lipase activity (HTGLA), and HDL-C subfractions were measured in 22 runners. LPLA was inversely related to triglyceride concentration ( r = −0.46, P < 0.01) and directly related to HDL-C ( r = 0.54, P < 0.01) and HDL 2-C ( r = 0.60, P < 0.01). In contrast, HTGLA was inversely related to HDL-C ( r = −0.36, P = 0.10) and HDLZ-C ( r = −0.39, P = 0.07). These data suggest that factors other than exercise training, diet and physical characteristics account for differences in HDL-C among distance runners, and that factors regulating HDL-C may do so by influencing tissue lipase activities.