Interstrand cross-links (ICLs) make up a unique class of DNA lesions in which both strands of the double helix are covalently joined, precluding strand opening during replication and transcription. The repair of DNA ICLs has become a focus of study since ICLs are recognized as the main cytotoxic lesion inflicted by an array of alkylating compounds used in cancer treatment. As is the case for double-strand breaks, a damage-free homologous copy is essential for the removal of ICLs in an error-free manner. However, recombination-independent mechanisms may exist to remove ICLs in an error-prone fashion. We have developed an in vivo reactivation assay that can be used to examine the removal of site-specific mitomycin C-mediated ICLs in mammalian cells. We found that the removal of the ICL from the reporter substrate could take place in the absence of undamaged homologous sequences in repair-proficient cells, suggesting a cross-link repair mechanism that is independent of homologous recombination. Systematic analysis of nucleotide excision repair mutants demonstrated the involvement of transcription-coupled nucleotide excision repair and a partial requirement for the lesion bypass DNA polymerase eta encoded by the human POLH gene. From these observations, we propose the existence of a recombination-independent and mutagenic repair pathway for the removal of ICLs in mammalian cells.