NADH-cytochrome b5 reductase (b5R) is a member of a flavoenzyme family of dehydrogenases-electron transferases that participates in the transfer of electrons from the NADH generated in glycolysis to cytochrome b5. b5R is involved in the steady-state reduction of methemoglobin to hemoglobin in erythrocytes and is also involved in lipid metabolism in other cell types. In a search for mutations of the b5R gene in two unrelated African-American families, a high-frequency polymorphism was detected in the propositi from both families, as well as unrelated normal controls, consisting of a C-to-G transversion in exon 5 that changes threonine to serine at codon 116 (T116S). This is the first polymorphism found in the b5R gene. Using allele-specific PCR on the two propositi, their family members, and unselected populations of African-American, Caucasian, Asian, Indo-Aryan, and Arabic individuals, the C/G polymorphism was found in 26 of 112. African-American chromosomes (allele frequency = 0.23), but not in 108 Caucasian, 46 Asian, 44 Indo-Aryan, or 14 Arabic chromosomes. In preliminary studies, this polymorphism did not correlate with b5R enzyme activity or cause any disease phenotype. It remains to be determined whether this African-specific polymorphism that apparently originated recently in human evolution provides any special survival advantage.