We have identified 12 individuals who are heterozygous for a chromosome with three alpha-globin genes. We determined the presence of the third alpha-globin locus by restriction endonuclease digestion and hybridization with alpha-globin cDNA probes. The three alpha-globin loci resided in an elongated fragment on digestion with EcoRI, BamHI, and Xba I, and the third locus was present in an additional 3.7-kilobase fragment on digestion with Hpa I, Sac I, and Bgl II. The locations of the restriction sites are compatible with a nonhomologous crossover mechanism producing the triple alpha loci. The frequency of the triple alpha loci was 0.0036 in American blacks and less than 0.004 in Sardinians, but was higher in Greek Crypriots (0.05). Individuals with five alpha-globin genes (alpha alpha/alpha alpha alpha) apparently showed no clinical or hematologic abnormalities.