The role of agouti signaling protein (ASIP) in human pigmentation pathways is not definitively understood although its murine homologue regulates, in part, pheomelanogenesis. We have reported an association of a polymorphism in the 3'-untranslated region of ASIP (g.8818A>G) with dark hair and eye color among a group of European-Americans (Am J Hum Genet 2002 March;70:770). Among 147 healthy control subjects, the frequency of the G-allele was 0.12. We hypothesized that this polymorphism would occur at different frequencies among different population groups. Using PCR-RFLP, we genotyped 25 East Asian, 86 African-American, and 207 West African individuals for the ASIP g.8818A>G polymorphism. The g.8818G-allele was present in the West African sample at a frequency of 0.80, in the African-American sample at a frequency of 0.62, and in the East Asian sample at 0.28. The difference in allele frequency among population groups was statistically significant (P < 0.0001). Although the effect of the g.8818A>G polymorphism upon ASIP function is unknown, the large difference in allele frequency between our West African and European-American sample populations lends support to the notion that this gene may be important in human pigmentation.