Herpesvirus saimiri, an oncogenic gamma herpesvirus of primates, is the only eukaryotic virus that carries the entire metabolic gene set for a complex biochemical synthesis. Every element of the thymidine synthesis gene cascade is present in the virus, and their function is probably related to the uniquely high A + T content of the genome. Although one member of the gene set, dihydrofolate reductase (DHFR), is mapped in a region required for oncogenesis, very little is known of the expression and function of this gene in transformed cells. We report the expression of the DHFR sequence on a novel, unique tricistronic transcript in virally transformed tumor cells. The DHFR sequence is the first open reading frame on a 5.3 kb minor transcript. Alpha-amanitine sensitivity indicates that it is an RNA polymerase II transcript, and since it is also polyadenylated it appears to be a functional, relatively unstable (half-life 3 hr) mRNA. Initiation of transcription uniquely overlaps with the HSUR3 small RNA gene. Expression of the small transcript appears to be alpha-amanitine resistant, implicating polymerase III transcription. Together with the remarkably low-level expression of HSUR3 in tumor cells, the data may indicate transcription interference between two different RNA polymerases, with unusual overlapping regulation and initiation.