Herpesvirus saimiri has been shown to possess a thymidylate synthase (TS) gene that is unusual in its transcriptional regulation. Although TS is believed to be required for viral DNA synthesis, the TS-specific 2.5-kb mRNA was found most abundantly during the late phases of asynchronous virus replication in permissive cultures. To study the kinetics of gene activation, the TS promoter and regulatory sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. No CAT expression or transcripts were found after transfection of fusion genes into permissive owl monkey kidney (OMK) cells. However, the promoter was strongly activated when CAT plasmids were cotransfected with intact herpesvirus saimiri virion DNA or were transferred to OMK cells that were lytically infected with herpesvirus saimiri or a related herpesvirus, herpesvirus ateles. CAT was expressed at reduced levels in cultures when viral DNA replication was inhibited by phosphonoacetic acid; this indicates that the gene is activated during the delayed-early phase. However, the highest amounts of mRNA were present in the late period of replication. Deletion analyses localized essential response elements for trans activation in the promoter upstream region between nucleotides -311 and -56; they consisted of related tandem repeats and perfect palindromes. A sequence with two overlapping palindromes of 16 and 18 bp was found to be a major target for activation of the herpesvirus saimiri TS promoter. These palindromes did not have any significant homologies with known sequences of herpesviruses or cellular DNA; the 18-bp palindrome had, however, a certain structural similarity with a conserved sequence of the E2-responsive cis sequence that is required for transcription regulation of early papillomavirus genes.