Defects in mismatch repair are associated with several types of cancer. It is also generally believed that environmental carcinogens are responsible for the initiation of cancers by the induction of mutations in critical genes. Prior genetic studies have suggested that the mismatch repair system can also recognize certain forms of DNA damage such as O6-methylguanine and UV photoproducts, and, therefore, mismatch repair may play a role in environmental agent-induced carcinogenesis. To examine this hypothesis, hMutSalpha, a heterodimer which consists of hMSH2 and GTBP and participates in strand-specific mismatch repair, was tested for its ability to recognize DNA containing a site-specific C8-guanine adduct of aminofluorene (AF) or N-acetyl-2-aminofluorene (AAF). We show here that hMutSalpha specifically binds to both AF and AAF adducts. This binding requires both hMSH2 and GTBP. Results from competition and titration experiments indicate that the binding efficiency of hMutSalpha to AF and AAF is about 60% of that to a G-T mismatch, but is at least 10-fold that to an otherwise identical homoduplex DNA without the chemical modification. The specific binding of AF and AAF adducts by hMutSalpha suggests that strand-specific mismatch repair is involved in processing DNA damage induced by environmental carcinogens.