The objective of this study was to provide population frequency distribution data for non-high-density lipoprotein (HDL) cholesterol (total cholesterol minus HDL cholesterol) concentrations and to evaluate whether differences exist by gender, ethnicity, or level of education. Serum levels of non-HDL cholesterol and sociodemographic characteristics were determined for 3,618 black, 3,528 Mexican-American, and 6,043 white women and men, aged >/=25 years, from a national cross-sectional survey of the US population (National Health And Nutrition Examination Survey III, 1988-1994). Age-adjusted non-HDL cholesterol concentrations were lower in women than men (154.1 vs 160.4 mg/dL, p <0.001). In women and men, age was positively associated with non-HDL cholesterol in the 25 to 64-year age range, and the slope of the association was steeper for women. For women and men >/=65 years, age was negatively associated with non-HDL cholesterol, and the slope of the association was steeper for men. Black women and men had lower non-HDL cholesterol levels than either Mexican-American or white women and men (women, p <0.02; men, p <0.001, for both ethnic contrasts). Women with less education had higher levels of non-HDL cholesterol than women with more education (p <0.01). These nationally representative population frequency distribution data provide non-HDL cholesterol reference levels for clinicians and investigators and indicate that there are significant variations in non-HDL cholesterol by gender, age, ethnicity, and level of education.