The centromeric regions of human and primate chromosomes are characterized by diverged subsets of tandemly repeated alpha-satellite DNA. Comparison of the alpha-satellites on known homologous chromosomes in human and chimpanzee provides insight into the very rapid evolution of satellite DNA sequences and the mechanisms that shape complex genomes. By using oligonucleotide primers specific for a conserved region of human alpha-satellite DNA, we have amplified a chromosome-specific alpha-satellite subset from the chimpanzee genome by the polymerase chain reaction. Fluorescence in situ hybridization showed that clones palphaPTR4N and palphaPTR4H are homologous to sequences at the centromere of the chimpanzee chromosome 4. This alpha-satellite subset is organized as a series of pentameric (higher-order) repeats, operationally defined by digestion of genomic DNA with HaeIII, MboI, RsaI, SstI, and XbaI. The lengths of four independent centromeric arrays measured by pulsed-field gel electrophoresis varied between 800 and 3,500 kb (mean = 1,850 kb, SD = 1,000 kb). Nucleotide sequence analysis demonstrated that chimpanzee chromosome 4 alpha-satellite is most closely related to the suprachromosomal subfamily II, which is evolutionarily different from the subfamily I to which the alpha-satellite on the homologous human chromosome 5 belongs. This implies that the human-chimpanzee sequence divergence has not arisen from a common ancestral alpha-satellite repeat(s) but instead represents concerted evolution of distinct repeats on homologous chromosomes.