The repair of damage induced by the alkylating antitumor drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was investigated using an in vitro excision repair system. Hamster cell extracts prepared from the parental CHO-9 cell line and the ERCC1 mutant 43-3B were both proficient in the repair of CCNU-induced damage. The in vitro repair of CCNU damage was faster than the repair of UV damage and plasmid substrates were rapidly and efficiently incised after incubation with either CHO-9 or 43-3B extracts. 7-Methylguanine (7-meG) and 3-methyladenine (3-meA) glycosylases were active to a similar extent in the CHO-9 and 43-3B extracts. The data indicate that most damage induced by CCNU is repaired via the ERCC1-independent base excision repair pathway, initiating with removal of chloroethylated and hydroxyethylated bases by N-glycosylases. Yet, the sensitive phenotype of 43-3B cells suggests that the ERCC1 gene product is required for the removal of a small subset of CCNU-induced lesions that are important for drug cytotoxicity.