The CHO UV-sensitive mutants UV24 and UV135 (complementation groups 3 and 5, respectively) are defective in nucleotide excision repair. After fusing each mutant with human lymphocytes, resistant hybrid clones showing genetic complementation were isolated by repeated exposure to UV radiation. Using a combination of isozyme markers, DNA probes, and cytogenetic methods to analyze the primary hybrids and their subclones, correction of the repair defect was shown to be correlated with the presence of a specific human chromosome in each case. Chromosome 2 corrected UV24, and the gene responsible was designated ERCC3. Line UV135 was corrected by human chromosome 13 and the gene designated ERCC5. The UV-sensitive mouse cell line, Q31, was shown not to complement UV135 and thus appears to be mutated in the same genetic locus (homologous to ERCC5) as UV135. Breakage of complementing chromosomes with retention of the genes correcting repair defects allowed the following provisional assignments: regional localization of ERCC5 to 13q14-q34, exclusion of ERCC3 from the region of chromosome 2 distal to p23, and relief of the ambiguity of ACP1 assignment (2p23 or 2p25) to 2p23 proximal to MDH1.