A 35-kbp region of genomic DNA encoding the human muscle phosphofructokinase (HPFK-M) gene including all of the coding exons (1-22) plus 2.2-kbp of 5'-flanking sequence has been cloned. The exon boundaries are the same as has been observed for the rabbit muscle phosphofructokinase (RPFK-M), the human liver phosphofructokinase (HPFK-L), and the mouse liver phosphofructokinase (MPFK-L) genes. Characterization of the structure of the HPFK-M gene and its transcript in Epstein-Barr virus transformed B-cell lines derived from patients with glycogen storage disease type VII (GSDVII or Tarui's disease) demonstrated that this single-copy gene encodes a normal sized 3.0-kb transcript in the four cases examined. This suggests the lesion in these cases represents either a point mutation or possibly a small insertion or deletion resulting in the synthesis of a defective HPFK-M protein. Analysis of the 5'-flanking region demonstrated the presence of a functional promoter located within 114 nucleotides of a proposed transcription initiation site. This promoter was active in the human cervical carcinoma cell line, HeLa S3, the dedifferentiated human hepatoma cell line, HepG2.1, and the mouse myoblast cell line, C2C12, suggesting this promoter has a broad cell-type specificity. In addition, from the known HPFK-M cDNA sequences, this observation indicates that the HPFK-M gene has a second promoter located upstream from the genomic region isolated in this study.