The murine DNA methyltransferase catalyzes the transfer of methyl groups from S-adenosylmethionine to cytosines within d(CpG) dinucleotides. The enzyme is necessary for normal embryonic development and is implicated in a number of important processes, including the control of gene expression and cancer, Metabolic labeling and high pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) were performed on DNA methyltransferase purified from murine erythroleukemia cells, Serine 514 was identified as a major phosphorylation site that lies in a domain required for targeting of the enzyme to the replication foci, These results present a potential mechanism for the regulation of DNA methylation. HPLC-ESI-MS peptide mapping data demonstrated that the purified murine DNA methyltransferase protein contains the N-terminal regions predicted by the recently revised 5' gene sequences (Yoder, J. A., Yen, R.-W. C., Vertino, P. M., Bestor, T. H., and Baylin, S. B. (1996) J. Biol, Chem, 271, 31092-31097), The evidence suggests a start of translation at the first predicted methionine, with no alternate translational start sites, Our peptide mapping results provide a more detailed structural characterization of the DNA methyltransferase that will facilitate future structure/function studies.