The binding of the transcription factors Sp1 and CTF immediately upstream from the TATA box of the Herpes simplex virus (type 1) thymidine kinase-coding gene (tk) facilitates efficient transcription of this gene in microinjected Xenopus laevis oocytes. To establish whether the presence of methylated CpG dinucleotides within the binding sites of these two factors affects transcription of the tk gene in vivo, we replaced a 33-bp promoter segment, consisting solely of the Sp1 and CTF binding sites, with synthetic oligodeoxynucleotide duplexes containing 5-methylcytosine residues at selected positions. We show that symmetrical methylation (modification of both strands) of any of the four CpGs within this promoter segment resulted in an approximately 20-fold reduction in the specific transcription of the tk gene in Xenopus oocytes, as shown by primer extension analysis of the isolated mRNA. As no other methylated CpG dinucleotides were present within the entire 9.2-kb vector, our results demonstrate that the presence of a single mCpG dinucleotide within the promoter region is sufficient for transcriptional inactivation of the tk gene. The possible mechanisms of this downregulation are discussed.