The p53 tumor suppressor protein is an important negative regulator of the G1 to S transition in mammalian cells. We have investigated the effect of p53 on the expression of the mouse thymidylate synthase (TS) gene, which normally increases as cells enter S phase. A luciferase indicator gene that was driven by the wild-type or various modified forms of the TATA-less mouse TS promoter was transiently cotransfected with a p53 expression plasmid into TS-deficient hamster V79 cells and the level of luciferase activity was determined. We found that wild-type p53 inhibited TS promoter activity by greater than 95% but had a strong stimulatory effect on an artificial promoter that contained multiple p53-binding sites. In contrast, an expression plasmid that encodes a mutant form of p53 or a wild-type retinoblastoma tumor suppressor protein had little effect on TS promoter activity. Deletion of sequences upstream or downstream of the TS essential promoter region, or inactivation of each of the known elements within the essential promoter region, had no effect on the ability of wild-type p53 to inhibit TS promoter activity. Our observations indicate that the inhibition of TS promoter activity by p53 is not due to the presence of a specific p53 negative response element in the TS promoter. Rather, it appears that p53 inhibits the TS promoter by sequestering ("squelching") one or more general transcription factors.