The expansion of (CTG)·(CAG) repeats has been associated with least 16 human diseases, including myotonic dystrophy type 1 (DM1) and is attributed to DNA slipped structure formation at the repeat tract during DNA metabolic processes. Towards identifying proteins that may be involved in processing these DNA structures, (CTG)·(CAG) slipped-structure binding activity was detected in HeLa cell extracts using gel shift assays. Binding activity was protein-dependent and partially resistant to SDS and heat denaturation, indicating that a multiprotein complex might be involved. Candidate proteins, including HMGB1, HMGB2 and nucleolin, were isolated by cell extract fractionation and affinity chromatography. Antibodies against these proteins caused a progressive decrease in the degree of electrophoretic shift, suggesting that HMGB1, HMGB2 and nucleolin may be components of a multiprotein slipped DNA structure binding complex. These results may have implications for the recognition and processing of slipped strand DNA structures and trinucleotide repeat instability.