While an unstable CTG triplet repeat expansion is responsible for myotonic dystrophy, the mechanism whereby this genetic defect induces the disease remains unknown. To detect proteins binding to CTG triplet repeats, we performed bandshift analysis using as probes double-stranded DNA fragments having CTG repeats [ds(CTG)6-10] and single-stranded oligonucleotides having CTG repeats ss(CTG)8 or RNA CUG triplet repeats (CUG)8. The source of protein was nuclear and cytoplasmic extracts of HeLa cells, fibroblasts and myotubes. Proteins binding to the double-stranded DNA repeat [ds(CTG)6-10], were inhibited by nonlabeled ds(CTG)6-10, but not by a non-specific DNA fragment (USF/AD-ML). Another protein binding to ssCTG probe and RNA CUG probe was inhibited by nonlabeled (CTG)8 and (CUG)8. Nonlabeled oligos with different triplet repeat sequences, ss(CAG)8 or ss(CGG)8, did not inhibit binding to the ss(CTG)8 probe. However, when labeled as probes, the (CAG)8 and (CGG)8 bound to proteins distinct from the CTG proteins and binding was inhibited by nonlabeled (CAG)8 or (CGG)8 respectively. The protein binding only to the RNA repeat (CUG)8 was inhibited by nonlabeled (CUG)8 but not by nonlabeled single- or double-stranded CTG repeats. Furthermore, the CUG-BP exhibited no binding to an RNA oligonucleotide of triplet repeats of the same length but having a different sequence, CGG. The CUG binding protein was localized to the cytoplasm, whereas dsDNA binding proteins were localized to the nuclear extract. Thus, several trinucleotide binding proteins exist and their specificity is determined by the triplet sequence. The novel protein, CUG-BP, is particularly interesting since it binds to triplet repeats known to be present in myotonin protein kinase mRNA which is responsible for myotonic dystrophy.