We report the cloning of the mouse pgk-1 gene encoding the somatic cell isoform of the enzyme phosphoglycerate kinase. The gene is contained within a 16-kb region of the X chromosome and is interrupted by at least ten introns. The promoter region of the pgk-1 gene is rich in G and C nucleotides and contains five copies of the hexadeoxynucleotide, GGGCGG, the potential binding site for the Sp 1 transcription factor, a CCAAT sequence, but no TATA box. This promoter functions following DNA-mediated transfection into mammalian cells. The promoter of the mouse pgk-1 gene is homologous to the human pgk-1 promoter. A number of conserved motifs in the promoter may indicate a significant role for these sequences in expression of the pgk-1 gene.