Using vector-CENP-B box polymerase chain reaction (PCR) we isolated and cloned from a human chromosome 21-specific plasmid library, a 1 kb DNA sequence, named pH21. In in situ hybridization experiments, pH21 hybridized, under high stringency conditions, to the centromeric region of all the human, chimpanzee, gorilla and orangutan chromosomes. On human chromosomes pH21 also identified non-centromeric sequences at 2q21 (locus D2F33S1) and 9q13 (locus D9F33S2). The possible derivation of these sequences from ancestral centromeres is discussed. Sequence analysis confirmed the alphoid nature of the whole pH21 insert.