DNA interstrand cross-links (ICLs) are complex DNA lesions generated by bifunctional alkylating agents, a class of compounds extensively used in cancer chemotherapy. Formation of an ICL covalently links the opposing strands of the double helix and results in severe disruptions of normal DNA functions, such as replication, transcription, and recombination. Because of the structural complexity, ICLs are most likely recognized by a variety of repair recognition proteins and processed through multiple mechanisms. To study the involvement of different repair pathways in ICL processing, we examined a variety of mammalian mutants with distinct DNA repair deficiencies. We found that the presence of ICLs induces frequent recombination between direct repeat sequences, suggesting that the single-strand annealing pathway may be an important mechanism for the removal of ICLs situated within direct repeats. Unlike recombination-independent ICL repair, ICL-induced single-strand annealing does not require the nucleotide excision repair (NER) mechanism. In cells defective in the mismatch repair protein Msh2, the level of recombination-independent ICL repair was significantly increased, suggesting that processing by the mismatch repair mechanism may lead to recombinational repair of ICLs. Our results suggest that removal of ICLs may involve two error-prone mechanisms depending on the sequence context of the cross-linked site.