SV40 DNA was methylated in vitro with prokaryotic or eukaryotic DNA cytosine-5-methyltransferases and the inhibition of transcription by methylation was studied in Xenopus oocytes. Methylation with the prokaryotic HhaI or HpaII methyltransferases did essentially not inhibit transcription of SV40. Methylation with a rat liver methyltransferase led only to minor inhibition of the SV40 early genes, but to a complete shut shut off of the SV40 late genes. Partial methylation showed that methylation of both, the regulatory region and the 5' end of the SV40 late genes, was necessary for the effect on transcription. The TK gene could be inactivated by eukaryotic methylation of either the promoter and the first 50 nucleotides of the gene or the 3' rest of the gene. Insertion of the SV40 enhancer, not containing methylatable CpGs, into the TK upstream region, had no influence on the inhibition of TK gene transcription by methylation.