Interstrand cross-links (ICL) in DNA arise from bifunctional alkylating agents, including nitrogen mustards, mitomycin C and psoralens. Such adducts prevent normal transcription or replication and are mutagenic. Therefore, cellular mechanisms for removing ICL damage are needed to maintain genome stability. Normal ICL repair requires the action of a number of genes, some specific for such damage. The yeast Snm1 protein is one such protein, but its function has been unknown. Incision for ICL repair is normal in mutants lacking Snm1, so it appears to act after the earliest steps. We have used recombinant SNM1 constructs in an Escherichia coli (E. coli) expression system to demonstrate that the yeast gene encodes a 5'-exonuclease. The exonuclease activity is required for Snm1 to be functional in ICL repair.