The article identifies and characterizes three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain (MBD). These proteins specifically bind methylated DNA in vitro and colocalize with methylated sequences in vivo, suggesting they are mediators of the biological consequences of DNA methylation. MBD2 and MBD4 bind methylated DNA in both mouse and human cells, while MBD3 does not bind methylated DNA in either context. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem (ES) cells, which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo, highlighting their potential role in mediating the effects of DNA methylation.The article identifies and characterizes three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain (MBD). These proteins specifically bind methylated DNA in vitro and colocalize with methylated sequences in vivo, suggesting they are mediators of the biological consequences of DNA methylation. MBD2 and MBD4 bind methylated DNA in both mouse and human cells, while MBD3 does not bind methylated DNA in either context. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem (ES) cells, which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo, highlighting their potential role in mediating the effects of DNA methylation.