An activity in nuclear extracts of S.cerevisiae binds specifically to heteroduplexes containing four to nine extra bases in one strand. The specificity of this activity (IMR, for insertion mismatch recognition) in band shift assays was confirmed by competition experiments. IMR is biochemically and genetically distinct from the MSH2 dependent, single base mismatch binding activity. The two activities migrate differently during electrophoresis, they are differentially competable and their spectra of mispair binding are distinct. Furthermore, IMR activity is observed in extracts from an msh2- msh3- msh4- strain. IMR exhibits specificity for insertion mispairs in two different sequence contexts. Binding is influenced by the structure of the mismatch since an insertion with a hairpin configuration is not recognized by this activity. IMR does not result from single-strand binding because single-stranded probes to not yield IMR complex and single-stranded competitors are unable to displace insertion heteroduplexes from the complex. Similar results with intrinsically bent duplexes make it unlikely that recognition is conferred by a bend alone. Heteroduplexes bound by IMR do not contain any obvious damage. These findings are consistent with the idea that yeast contains a distinct recognition factor, IMR that is specific for insertion/deletion mismatches.