Apolipoprotein (apo) C-I is a constituent of triglyceride-rich lipoproteins (TGRL) that interferes with their hepatic clearance. Functional polymorphism in the apoC-I gene has not been established. We determined that an Hpa I site variably present at −317 relative to the apoC-I gene is produced by a 4-bp CGTT insertion. The apoC-I Hpa I alleles showed an ethnically distinct pattern of linkage disequilibrium with the alleles of the adjacent apoE gene. The frequency of apoC-I Hpa I-positive (H2) with apoE ε2 was 0.98, without significant ethnic difference. In contrast, the frequency of H2 with apoE ε4 was 0.85 in European-Americans but only 0.55 in African-Americans (P < 0.001). The frequency of H2 with apoE ε3 was 0.02 in European-Americans and 0.08 in African-Americans (P < 0.001). African-American apoE ε3/ε3 carriers of apoC-I H2 had 19% lower fasting triglyceride levels than H1 homozygotes (P = 0.03) along with 18% higher HDL-cholesterol levels (P = 0.02). ApoB levels were 21% lower (P = 0.002). H2-allelic reporter-gene constructions showed 50% higher expression in transient transfection studies. We localized the source of this difference in expression to the CGTT insertion itself. Deletion studies of the H1 allele showed a negative transcriptional effect of the polymorphic region. An H1 oligodeoxynucleotide showed specific binding of a hepatomacell nuclear protein not evident with an H2 oligodeoxynucleotide. The H2 sequence may decrease the binding of a negatively acting transcription factor, leading to overexpression of apoC-I. This may produce a functional effect on lipoprotein levels but confirmation is needed in other populations.—Xu, Y., L. Berglund, R. Ramakrishnan, R. Mayeux, C. Ngai, S. Holleran, B. Tycko, T. Leff, and N. S. Shachter. A common Hpa I RFLP of apolipoprotein C-I increases gene transcription and exhibits an ethnically distinct pattern of linkage disequilibrium with the alleles of apolipoprotein E. J. Lipid Res. 1999. 40: 50–58.