The human excision-repair gene ERCC3 was cloned after DNA-mediated gene transfer to the uv-sensitive Chinese hamster ovary mutant cell line 27-1, a member of complementation group 3 of the excision-defective rodent cell lines. The ERCC3 gene specifically corrects the DNA repair defect of xeroderma pigmentosum (XP) complementation group B, which displays the clinical symptoms of XP as well as of another rare excision-repair disorder, Cockayne syndrome. The gene encodes a presumed DNA and chromatin binding helicase, involved in early steps of the excision-repair pathway. ERCC3 was previously assigned to human chromosome 2 (L.H. Thompson, A.V. Carrano, K. Sato, E.P. Salazar, B.F. White, S.A. Stewart, J.L. Minkler, and M.J. Siciliano (1987) Somat. Cell Genet. 13: 539-551). Here we report its subchromosomal localization in the q21 region of chromosome 2 via somatic cell hybrids containing a translocated chromosome 2 and in situ hybridization with fluorescently labeled ERCC3 probes.