Human glycophorin A, B, and E genes are homologous from the 5'-flanking region to 1 kilobase downstream from the exon encoding the transmembrane region. Analysis of human Alu sequences at the transition site from the homologous to nonhomologous region suggested that the GPA gene most closely resembles the ancestral gene, whereas GPB and GPE genes arose by homologous recombination within the Alu repetitive sequence, and acquired 3' sequences from an unrelated gene (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623; Kudo, S., and Fukuda, M. (1990) J. Biol. Chem. 265, 1102-1110). To understand glycophorin gene evolution in primate phylogeny, transmembrane and Alu regions of several primate genomes were amplified by the polymerase chain reaction and their sequences were analyzed. These studies revealed that the GPA gene was present in all primates studied, and the GPB gene was present in pygmy chimpanzee, chimpanzee, and gorilla, but absent from orangutan and gibbon. GPE gene was present in all species with a GPB gene, but was detected in only 7 out of 16 gorillas. The 24-base pair insertion sequence found in the transmembrane exon of the human GPE gene was shown to be derived from the ancestral GPB gene and was inserted into the ancestral GPE gene prior to gorilla divergence. The recombination site in the GPA gene was confirmed to be within an Alu repetitive sequence. We conclude that GPB and GPE genes arose from an ancestral GPA gene via two gene duplications occurring during primate evolution, prior to gorilla divergence.