The Sak gene encodes a serine/threonine kinase, which is a member of the Polo family of mitotic regulators. Sak transcripts are present in S/G2/M phase cells, and in proliferating cell layers of the mouse embryo and adult tissues. In this report, we have characterized the murine Sak gene structure, the Sak chromosomal location, and identified the promoter. The murine Sak gene is located on the proximal arm of mouse chromosome 13, as determined by RFLP analysis. The murine gene comprises 15 coding exons spanning 16kb of genomic sequence, and encodes two alternately spliced transcripts. Sak-a, the predominant transcript, is encoded by 15 exons, while early termination of transcription and alternative splicing at exons 5 and 6 results in Sak-b. This truncated transcript encodes the complete kinase domain and a carboxyl end translated from 147bp of sequence contiguous with exon 5. Human Sak-a (Stk18) cDNA is reported to contain an insertion of sequence corresponding to the mouse Sak-b tail. Primer extension analysis of murine Sak revealed one major transcription start site at position -303bp relative to the start of translation. A genomic fragment of 3.5kb located 5' of the Sak transcriptional start drives expression of a luciferase-reporter gene in CHO and GC1-SPG cells in an orientation-dependent fashion. Using various Sak promoter/luciferase constructs, the core promoter region required for expression was located within 400bp of the message Cap site, and sequence further 5' strongly suppressed transcription.