We encountered a homozygous -16C>G mutation in cis at identical positions in the promoters of both human gamma-globin genes in a subject who was also homozygous for Hemoglobin C (HbC). Subsequent analysis of normal control individuals of African American ancestry revealed that both mutations were always present in cis with an allelic frequency of 3%. Furthermore, 10 out of 11 HbC subjects carried the -16C>G sequence variations, suggesting an association with HbC. The -16C>G mutation disrupts a putative CACCC box positioned between the TATA box and the transcriptional start site. However, the absence of high levels of HbF in HbC subjects homozygous and heterozygous for the -16C>G sequence variation suggested no effect of this mutation on gamma-globin gene expression in the adult stage of development. Further functional characterization by means of transient transfections in human erythroleukemic K562 cells showed that the -16C>G promoter sequence variation did not have an effect on gamma-globin expression in the fetal stage of development either. We therefore conclude that the -16C>G gamma-globin sequence variations are not beneficial to the clinical phenotype of HbC. The unique concurrent presence of this non-functional sequence variation is likely the result of a gene conversion event, and supports the concept of sequence homogenization between the two human gamma-globin genes.