Genetic variation in the human cholesteryl ester transfer protein (CETP) promoter has been shown to be associated with high-density lipoprotein cholesterol (HDL-C) levels and cardiovascular disease. Some of this variation occurs in Sp1/Sp3 binding sites in the proximal promoter. We find that both the known promoter polymorphism at -629 and the previously uncharacterized polymorphism at -38 are associated with HDL-C levels in vivo and affect transcription in vitro. While the -629 polymorphism is common in all ethnic groups, the -38 polymorphism is found at significant levels (6.4%) only among African Americans. Those homozygous for the less common -38A allele have higher HDL-C levels than those with the more frequent -38G allele. This association was found in a population of African Americans at risk of cardiovascular disease and then replicated in a different population chosen from among patients with extremes of HDL-C. When studied in vitro, the most transcriptionally active allele (-629C/-38G) yields 51% more reporter protein than the least active allele (-629A/-38A) in HepG2 cells. These transcriptional effects reflect the projected impact of increased CETP expression on HDL-C phenotypes seen in vivo.