We used nondenaturing polyacrylamide gradient gel electrophoresis to examine the associations of age, adiposity, alcohol intake, and exogenous estrogen with high-density lipoprotein (HDL) subclasses in 427 members of 51 principally Mormon kindreds. The absorbency of protein stain was used as an index of mass concentrations at intervals of 0.01 nm within five HDL subclasses: HDLj,. (7.2 to 7.8 nm), HDL, b (7.8 8.2 nm), HDL, (8.2 to 8.8 nm), HDL 2a (8.8 to 9.7 nm), and HDL 2b (9.7 12 nm). Age and alcohol intake were obtained from questionnaires, and body mass index was computed from clinic measurements as weight (kg)/height (m) 2. The results suggest that HDL, concentrations were higher after menopause than before. Adult men (>18 years old) had significantly higher HDL$ and HDL, b and significantly lower HDL 2b HDL?* levels than younger boys. Compared with the women, adult men had higher levels of HDI$. and HDL, b and lower levels of HDL 2b, HDL 2a and larger-diameter HDL Ja particles. There were no significant differences between the HDL profiles of women and younger boys, suggesting that the divergence in HDL occurs during puberty. Eighty-eight percent of the increase in HDL associated with estrogen replacement in postmenopausal women occurred within HDL, and HDL 2a. Reported alcohol intake in adult men correlated with two HDL regions: one within the HDL 2b region and a second within the HDL, region, whereas in women the positive correlation between alcohol and HDL levels was within the HDL 2b region only. In both men and premenopausal adult women, increasing levels of body mass index were associated with higher levels of HDL$ and lower levels of HDL 2b. Statistical adjustment for HDL cholesterol levels eliminated the significant relations of alcohol with HDL subclasses in both men and women. The adjustment did not eliminate the significant relations of age and body mass index to HDL$ and HDL;,. Thus, gradient gel electrophoresis of HDL subclasses appears to identify important physiological relations that are independent of HDL cholesterol levels.