By hybridizing total human DNA with probes derived from the extrachromosomal circular DNA fraction of cultured cells, we detected a human multisequence family, called chAB4, previously unknown. Approximately 50 copies of this sequence are located in the haploid human genome. The repetition units of chAB4 are 35 kb long and the units are tandemly arranged. DNA sequence analysis of parts of the chAB4 unit revealed no direct evidence for a possible function of the family, but possibly chAB4 harbors a gene. Family members are located on human chromosomes 1, 3, and 9 and on the short arms of chromosomes 13-15, 21, and 22. Therefore, in addition to the rDNA, chAB4 is the second class of clustered repetitive sequences with a relatively long repetition unit localized on the short arms of all acrocentric chromosomes. Some evolutionary aspects arising from the structure of chAB4, the established parts of its DNA sequences, and the chromosomal localization of this new multisequence family are discussed. © 1991.