We detected a novel nuclear protein, MMBP-3, that bound to the c-Myc binding motif (CACGTG) in which deoxycytidine in the CpG sequence was methylated. MMBP-3 was partially purified by chromatography on heparin-agarose and hydroxyapatite, followed by affinity adsorption to a matrix coupled to the methylated binding motif. Its binding to the methylated c-Myc binding motif was specific, although it also recognized the unmethylated motif weakly. MMBP-3 was further found to recognize only one of two differently hemimethylated forms of the double-stranded c-Myc binding motif. MMBP-3 activity was detected in proliferating C2C12 and C3H/10T1/2 cells, and down regulated when the growth of these cells was inhibited. We propose that MMBP-3 plays a role in regulating the c-Myc function by recognizing the methylation state of the c-Myc binding motif in a growth-dependent manner.