The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.