Cytosine DNA methyltransferases (MTases) first recognize specific nucleotide sequences and then transfer a methyl group from S-adenosylmethionine to cytosine. This division of function is reflected in five highly conserved motifs shared by cytosine MTases. The region containing the first four motifs is responsible for the catalytic function whereas the region containing the fifth motif V provides specificity of binding to DNA. In at least one case, two separate proteins, one containing the first four motifs and the second containing the last motif combine to provide full functional activity. In the frog virus 3 (FV3) genome we have identified an open reading frame (ORF) whose deduced amino acid (aa) sequence contains motifs characteristic of prokaryotic as well as eukaryotic MTases. The ORF consists of 642 bp which codes for a protein of 214 aa with a predicted molecular mass of 24.8 kDa. This ORF contains the first four highly conserved motifs of cytosine MTases but the fifth motif, responsible for DNA binding specificity, is missing. Presumably, FV3 MTase is composed of two subunits. Northern blot analysis showed that the putative MTase ORF is transcribed into two transcripts belonging to the delayed-early class of FV3 messages. These two transcripts appear to be initiated at two different start sites but terminate in the same 3' region of the gene. The transcription start sites are not preceded by any known promoter sequences, but two regions of hyphenated dyad symmetry are present at the 3' end of the message. A protein with a molecular mass of approximately 28 kDa was synthesized by a rabbit reticulocyte lysate programmed with capped runoff transcripts from the cloned gene, suggesting that the ORF can be transcribed into a message coding for a viral protein. Overall, our results suggest that we have identified a gene for a subunit of MTase in the FV3 genome.