Apolipoprotein A-I (apo A-I) is the most abundant protein in high-density lipoprotein (HDL) particles, and it plays an important role in HDL metabolism. Both apo A-I and HDL cholesterol (HDL-C) levels are inversely associated with risk of cardiovascular disease. Segregation analyses suggest apo A-I levels are under the control of one or more major loci. Since HDL particles are heterogeneous in their composition and size, genetic influence on its subfractions (i.e., HDL2 and HDL3) could vary. A previous report showed evidence of a major locus controlling HDL3-C levels in a subset of the current study population. Because quantitative trait loci involved in complex diseases are likely to have pleiotropic effects on several related traits, it is possible to have a common major gene involved in regulating apo A-I and HDL3-C levels. We performed a bivariate segregation analysis of apo A-I and HDL3-C levels in 1,006 individuals from 137 families ascertained through probands undergoing elective, diagnostic coronary angiography at the Johns Hopkins Hospital. The results showed significant genetic correlation between these two traits, but the hypothesis of a common major gene was rejected. Bivariate segregation analysis favored a model with two genes controlling apo A-I and a third gene independently controlling HDL3-C, and the genetic correlation between these two traits is due to residual additive polygenes. Overall, results from this study suggest that there are distinct genetic mechanisms for apo A-I and HDL3-C levels. Future studies, especially linkage analysis, should consider distinct genetic mechanisms and multiple major gene loci.