Numerous rodent cell lines exist that have defects in nucleotide excision repair of DNA caused by alterations in genes that fall into 10 different complementation groups. The precise roles in the repair of these genes are unknown. We report here that extracts from Chinese hamster ovary cells of excision repair-defective complementation groups 1 and 3 are defective in DNA excision repair in a cell-free system. In vitro complementation can be achieved by mixing extracts from the two groups with one another. In addition, extracts from a human cell line representing xeroderma pigmentosum complementation group B could complement rodent complementation group 1 extracts, but not group 3 extracts. This is consistent with an identity of the ERCC-3 and xeroderma pigmentosum group B genes. Cellular evidence points toward a defect in the incision of damaged DNA in group 1 and 3 mutants. Since the ERCC-1 and ERCC-3 proteins are required for the in vitro reaction, it appears that both gene products are directly involved in the enzymatic incision of damaged DNA, or in preincision reactions. The experiments reported here provide the biochemical basis of an approach to analyze the function of these nucleotide excision repair proteins.