Like vertebrates, the genome of Drosophila melanogaster also contains methylated cytosines. However, the enzyme(s) responsible for this methylation has been elusive. By DNA transfection and sodium bisulfite sequencing, we show here that overexpression of dDnmt2, which is the only expressed and cloned Drosophila protein consisting of motifs conserved among the DNA cytosine methyltransferases, results in genomic DNA methylation of Drosophila S2 cells. The data provide the first evidence for dDnmt2 being one candidate gene encoding the Drosophila DNA methyltransferase(s).