A number of clones that specifically hybridize to the human hsp60 cDNA (chaperonin protein; GroEL homolog) were isolated from human and Chinese hamster ovary cell genomic libraries. DNA sequence analysis shows that one of these clones, pGem-10, is completely homologous to the human hsp60 cDNA (in both coding and noncoding regions) with no intervening sequences. The other human clones analyzed were all nonfunctional pseudogenes containing numerous small additions, deletions, and base substitutions, but no introns. On the basis of sequence data, six different hsp60 pseudogenes were identified in human cells. In addition, we also cloned and completely sequenced a genomic clone from CHO cells. This clone, which was also a pseudogene, contained a small 87-nucleotide intron near the 3' end. Southern blot analysis of human, mouse, and Chinese hamster DNA, digested with unique restriction enzymes (no sites in cDNA), indicates the presence of about 8-12 genes for hsp60 in the vertebrate genomes. The sequence data, however, suggest that most of these genes, except one (per haploid genome), are likely to be nonfunctional pseudogenes.