Biochemical measures of nutrients or other dietary constituents can be an important component of nutritional assessment and monitoring. However, accurate interpretation of the nutrient concentration is dependent on knowledge of the determinants of the body pool measured. The purpose of this study was to identify the determinants of serum carotenoid and fat-soluble vitamin concentrations in a large, community-based sample (n = 1042). Multiple linear regression analysis was used to examine effects of demographic characteristics (age, sex, race/ethnicity, education), health-related behavior (exercise, sun exposure, smoking, alcohol consumption), and intake (diet, supplements) on serum retinol, 25-hydroxyvitamin D, alpha-tocopherol, phylloquinone, and carotenoid concentrations. Age, sex, race/ethnicity, vitamin A intake, and alcohol consumption were found to be determinants of serum retinol concentration. Race/ethnicity, vitamin D intake, body mass index, smoking status, and sun exposure were determinants of serum 25-hydroxyvitamin D concentration. Determinants of serum alpha-tocopherol were age, sex, race/ethnicity, alpha-tocopherol intake, serum cholesterol, percentage of energy from fat (inversely related), supplement use, and body mass index. Age, sex, phylloquinone intake, serum triglycerides, and supplement use were determinants of serum phylloquinone concentration. Primary determinants of serum carotenoids were age, sex, race/ethnicity, carotenoid intake, serum cholesterol, alcohol consumption, body mass index, and smoking status. Overall, the demographic, dietary, and other lifestyle factors explained little of the variability in serum concentrations of retinol (R2 = 0.20), 25-hydroxyvitamin D (R2 = 0.24), and the carotenoids (R2 = 0.15-0.26); only modest amounts of the variability in serum phylloquinone concentration (R2 = 0.40); and more substantial amounts of the variability in serum alpha-tocopherol concentration (R2 = 0.62).