Myotonic dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the RNA splicing factor muscleblind-like (MBNL) is sequestered to the expanded CUG or CCUG RNAs. Mislocalization of MBNL results in missplicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. We demonstrate that MBNL can bind short structured CUG and CCUG repeats with high affinity and specificity. Only 6 base pairs are necessary for MBNL binding: two pyrimidine mismatches and four guanosine–cytosine base pairs in a stem. MBNL also has a preference for pyrimidine mismatches, but many other mismatches are tolerated with decreased affinity. We also demonstrate that MBNL binds the helical region of a stem–loop in the endogenous pre-mRNA target, the cardiac troponin T (cTNT) pre-mRNA. The stem–loop contains two mismatches and resembles both CUG and CCUG repeats. In vivo splicing results indicate that MBNL-regulated splicing is dependent upon the formation of stem–loops recognized by MBNL. These results suggest that MBNL may bind all of its RNA substrates, both normal and pathogenic, as structured stem–loops containing pyrimidine mismatches.