The Gapd-s gene encodes an isoform of the glyceraldehyde 3-phosphate dehydrogenase enzyme expressed only in post-meiotic spermatogenic cells. Two clones containing the Gapd-s gene were isolated from a mouse genomic library. Sequencing and restriction enzyme analysis demonstrated that this single-copy gene contains 11 exons and spans 9596 base pairs. The locations of Gapd-s exons and introns are conserved when compared to the corresponding portions of the chicken and human somatic Gapd genes. The promoter region contains no TATA box, although there is a potential SP1 recognition site within exon 1. Like other TATA-less genes, primer extension analysis reveals some heterogeneity in the site of transcription initiation with Gapd-s transcripts initiating from three discrete sites. Northern analysis demonstrated that a 1.5-kb Gapd-s mRNA is expressed in the testis in at least three mammalian orders, indicating that the Gapd-s gene appeared early in mammalian evolution. Using GAPD-deficient bacteria, mouse GAPD-S was shown to be capable of functioning as a glycolytic enzyme. Since GAPD has been proposed to be a key enzyme regulating glycolysis in spermatogenic cells, GAPD-S may represent a potential target for toxicological or contraceptive agents affecting fertility by interfering with glycolysis.