The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD-DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16(INK4a) genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing.