To study apolipoprotein A-II, a simple, precise, and accurate immunodiffusion assay was developed and applied in a population sample of industrial employees. Apolipoprotein A-II (A-II) did not increase with age in men (r = −0.20, n = 172), but showed a slight increase with age in women (0.1 mg/dl per yr, r = 0.20, n = 188). A-II correlated significantly with apolipoprotein A-I (A-I) (r = 0.71) and high density lipoprotein (HDL) cholesterol (men, r = 0.64; women, r = 0.49). The A-I/A-II ratio was significantly related to HDL cholesterol (men, r = 0.29; women, r = 0.44). Women on no medication (n = 92) had A-II levels similar to men (34±5 and 33±5 mg/dl, mean±SD, respectively), whereas women on oral contraceptives or estrogens had significantly higher levels (39±6 mg/dl, n = 75, P < 0.01). The plasma A-I/A-II weight ratio was 3.6±0.4 for men and 3.8±0.5 for women. In the d = 1.10-1.21 subfraction, both males and females had similar A-I, A-II, and HDL cholesterol levels (men: mean, 97, 27, and 32 mg/dl, respectively; women: mean, 104, 28, and 36 mg/dl, respectively). Women had approximately twice the amount of A-I, A-II, and HDL cholesterol than men in the d = 1.063-1.10 fraction (men: mean, 10, 2, and 10 mg/dl, respectively; women: mean, 24, 4, and 19 mg/dl, respectively). The A-I/A-II weight ratio in the d = 1.063-1.10 fraction (men, 5.1±0.7; women, 6.1±1.3) was significantly greater (P < 0.01) than that in the d = 1.10-1.21 fraction (men, 3.7±0.2; women, 3.8±0.2). Furthermore, the weight ratio of cholesterol to total apoprotein A in the d = 1.063-1.10 fraction (men, 0.75±0.09; women, 0.67±0.05) was significantly higher (P < 0.01) than that found in the d = 1.10-1.21 fraction (men, 0.26±0.04, women, 0.28±0.05). Thus, the compositions of HDL hydrated density subclasses are significantly different from each other. These results suggest that the differences in HDL between men and women are due primarily to differences in the relative proportions of HDL subclasses rather than to the intrinsic differences in HDL structure.