We previously reported a compound heterozygote [T(-39)C/T(-93)G] in the human lipoprotein lipase (LPL) gene promoter in one out of 19 patients with familial combined hyperlipidemia (FCHL) and reduced post-heparin plasma LPL levels. The T(-39)C substitution resulted in 85% decrease in LPL promoter activity. Further screening of Caucasian patients with FCHL, coronary artery disease (CAD), and of unselected Caucasian subjects revealed four additional LPL promoter variants. Among the same 19 FCHL patients with reduced LPL levels, we found one heterozygote for a G(-53)C substitution. Among 115 CAD patients, we found five heterozygotes and one homozygote for the T(-93)G substitution and one heterozygote for a CC insertion between +13 and +19 of the 5' untranslated region. In a group of 183 unselected subjects, three heterozygotes with the T(-93)G substitution were found. The G(-53)C substitution led to approximately 70-75% decrease in promoter activity as assayed by transient transfections of THP-1 (macrophage-like) and C2C12 (myotube-like) cells. The T(-93)G substitution resulted in reduction of promoter activity by approximately 40-50%. The CC insertion between +13 and +19 caused a decrease in promoter activity by 20% in THP-1 and 50% in C2C12. Substitutions at -79 and -95, which had no effect on promoter function, were also discovered in the population samples studied. The finding of two promoter mutations (-39 and -53) among 19 FCHL patients with diminished LPL, but not among the other groups of subjects, suggests a potential role of regulatory mutations of the LPL gene in the development of dyslipidemia in FCHL.