Besides modulating specific DNA-protein interactions, methylated cytosine, frequently referred to as the fifth base of the genome, also influences DNA structure, recombination, transposition, repair, transcription, imprinting, and mutagenesis. DNA (cytosine-5-)-methyl-transferase catalyzes cytosine methylation in eukaryotes. We have cloned and expressed this enzyme in Escherichia coli, purified it to apparent homogeneity, characterized its properties, and we have shown that it hemimethylates DNA. The cDNA for murine maintenance methyltransferase was reconstructed and cloned for direct expression in native form. Immunoblotting revealed a unique protein (M(r) = 190,000) not present in control cells. The mostly soluble overexpressed protein was purified by DEAE, Sephadex, and DNA cellulose chromatography. Peak methylating activity correlated with methyltransferase immunoblots. The purified enzyme preferentially transferred radioactive methyl moieties to hemimethylated DNA in assays and on autoradiograms. All of the examined properties of the purified recombinant DNA methyltransferase are consistent with the enzyme purified from mammalian cells. Further characterization revealed enhanced in vitro methylation of premethylated oligodeoxynucleotides. The cloning of hemimethyltransferase in E. coli should allow facilitated structure-function mutational analysis of this enzyme, studies of its biological effects in prokaryotes, and potential large scale methyltransferase production for crystallography, and it may have broad applications in maintaining the native methylated state of cloned DNA.