The promoter of the mouse thymidylate synthase (TS) gene lacks both a TATAA box and an initiator element and directs transcriptional initiation at multiple sites over a 90 nucleotide initiation window. Earlier studies defined an essential region near the 5' end of the initiation window that is required for promoter activity. The essential region contains possible binding sites for Sp1 and Ets transcription factors. In the present study we show that this essential region stimulates transcription with approximately equal strength in both directions. Transcription is initiated over a broad initiation window in the reverse direction. The same elements are important for the reverse promoter and for the normal TS promoter. Sequences upstream of the essential region partially suppress expression in the reverse direction. The TS 5' flanking region, in either the normal or inverted orientation, directs S phase-specific expression of a TS minigene. This raises the possibility that an upstream gene and the TS gene may be coordinately induced at the G1/S phase boundary by a common set of control elements.