Genetic factors strongly influence interindividual variation in plasma high density lipoprotein cholesterol (HDL-C) levels, but the specific genetic polymorphisms that confer heritable variation in HDL-C levels have not been identified. In this study we examined the relationship between polymorphism in LIPC , the gene encoding hepatic lipase, and plasma HDL-C concentrations using a sequential approach comprising linkage analysis, DNA sequencing, and association studies. Linkage studies in 1465 American white subjects from 218 nuclear families indicated that allelic variation at, or closely linked to, the hepatic lipase gene accounts for a significant fraction (≈25%) of the variation in plasma HDL-C concentrations. The hepatic lipase gene was then sequenced in selected individuals, and four novel polymorphisms were identified in the 5′ flanking region of the gene. These polymorphisms were in complete linkage disequilibrium and thus identified a single novel allele. Association studies indicated that heterozygosity for the rare allele was associated with modestly increased concentrations of plasma HDL-C (41 ± 11 vs. 37 ± 10 mg/dl, P < 0.05) and apolipoprotein AI in men (131 ± 23 vs. 122 ± 21 mg/dl, P < 0.05) but not in women. Homozygosity for the rare allele was associated with markedly higher plasma HDL-C (63 ± 3 mg/dl) and apolipoprotein AI (153 ± 9 mg/dl) concentrations in men. The results of the association study were replicated in a second, independently ascertained sample. Taken together, the results of the linkage and association studies provide strong evidence that genetic variation in hepatic lipase activity is a major determinant of plasma HDL-C levels.