DNA cytosine 5-methyltransferase has been extensively purified (about 2600-fold) from the soft tissue of human placenta by chromatography on DEAE-cellulose and hydroxyapatite, and by an affinity step on agarose-immobilized S-adenosylhomocysteine. The isolated enzyme has a molecular weight of 135,000 and methylates DNA from various sources in native and heat-denatured forms. The synthetic copolymer poly(dG-dC) . poly(dG-dC) is methylated in B- and Z-conformation to about the same extent. DNA containing hemimethylated sites was isolated from P815 cells grown in the presence of 5-azacytidine. This P815 DNA was used to measure the "maintenance' DNA methylase activity, whereas 5-methylcytosine-free procaryotic DNA served as a substrate for the "de novo' DNA methylase activity in our enzyme preparation. The crude extract as well as the highly purified DNA methylase are capable of transferring methyl groups to these two types of substrate. The fact that both types of activity co-chromatograph during the isolation procedure suggests that one enzyme molecule may exercise both the "maintenance' and "de novo' activity.