The effects of DNA methylation on transcription and chromatin structure require that nuclear factors be able to distinguish methylated and nonmethylated DNA. We describe a methyl-CpG binding protein (MeCP) that complexes with a variety of unrelated DNA sequences when they are methylated at CpG. Fifteen or more symmetrically methylated CpG moieties per molecule are required for strong binding under our conditions. Competition experiments show that vertebrate DNAs bind to MeCP, whereas naturally nonmethylated genomes or cloned vertebrate genomes do not bind. Cross-linking experiments detect a 120 kd protein that correlates stringently with MeCP activity. Species and tissue comparisons show that MeCP is widely distributed in mammals except in embryonal carcinoma cell lines, which have very low levels.