The steric course of methyl group transfer catalyzed by two DNA methylases, HhaI methylase, a DNA (cytosine-5)-methyltransferase, and EcoRI methylase, which methylates at N6 of adenosine, has been studied with (methyl-R)- and (methyl-S)-[methyl-2H1,3H]adenosylmethionine as the methyl donor, using as substrates poly-d(GC) (HhaI) and the dodecamer oligonucleotide duplex d(CGCGAATTCGCG) (EcoRI), respectively. The methylated nucleotides were degraded to convert the chiral methyl groups into acetic acid for configurational analysis. It was found that both enzymatic reactions proceed with inversion of configuration of the methyl group.