Familial combined hyperlipidemia (FCH) is a common genetic lipid disorder in Western societies. In a recent report (Dallinga-Thie, G.M., X.D. Bu, M. van Linde-Sibenius Trip, J.I. Rotter, A.J. Lusis, and T.W.A. de Bruin. J. Lipid Res., 1996, 36:136-147) we have studied three restriction enzyme polymorphisms: XmnI, and MspI sites 5' of the apo AI gene and SstI site in the 3' untranslated region of exon 4 of the apo CIII gene in 18 FCH pedigrees, including 18 probands, 178 hyperlipidemic relatives, 210 normolipidemic relatives, and 176 spouses. DNA variations in the apo AI-CIII-AIV gene cluster had a modifying effect on plasma triglycerides, LDL cholesterol, and apolipoprotein CIII levels. In this study, combinations of haplotypes were analyzed to further characterize their interactions and effect on the expression of severe hyperlipidemia in FCH subjects. A specific combination of haplotypes with one chromosome carrying the X1M1S2 (1-1-2) haplotype and the other the X2M2S1 haplotype (2-2-1) was significantly more frequent in hyperlipidemic relatives (6%) than in normolipidemic relatives (3%) and spouses (0.5%). Associated with this combination of haplotypes were significantly elevated plasma cholesterol (P < 0.0001), triglycerides (P < 0.0001), and apo CIII (P < 0.001) levels when compared to the wild type combination of haplotypes 1-1-1/1-1-1. The only spouse with this specific combination of haplotypes showed a severe hyperlipidemic phenotype, similar to FCH. Furthermore, nonparametric sibpair linkage analysis revealed significant linkage between these markers in the gene cluster and the FCH phenotype (MspI P = 0.0088, SstI P = 0.044, and XMS haplotype P = 0.037). The present findings confirm that the apo AI-CIII-IV gene cluster contributes to the FCH phenotype, but this contribution is genetically complex. An epistatic interaction between different haplotypes of the gene cluster was demonstrated. The S2 allele on one haplotype was synergistic to the X2M2 allele on the other haplotype in its hyperlipidemic effect. Therefore, two different susceptibility loci exist in the gene cluster, demonstrating the paradigm of complex genetic contribution to FCH.