The genomic DNA encoding human muscle phosphofructokinase (HPFK-M) exons VII to X has been cloned and the coding regions have been sequenced. The intron/exon boundaries are located at the same positions as those identified for the rabbit phosphofructokinase-M gene (Lee, C. -P., Kao, M. -C., French, B. A., Putney, S. D., and Chang, S. H. (1987) J. Biol. Chem. 262, 4195-4199). A HPFK-M cDNA clone lacking the sequences corresponding to exon IX was isolated from a human fibroblast (IMR-90) library, suggesting that the HPFK-M transcript may be alternatively spliced. Exon IX is 93 nucleotides in length, and the absence of this sequence from the HPFK-M transcript would generate an RNA coding for a HPFK-M-related polypeptide lacking 31 amino acids compared with the HPFK-M polypeptide. HPFK-M transcripts approximately 3.0 kilobases in length are expressed in a tissue-specific manner with high levels in cell lines and skeltal muscle tissue and very low levels in peripheral blood mononuclear cells and liver tissue. Characterization of the structure of these HPFK-M transcripts by nuclease S1 and polymerase chain reaction analysis demonstrated that all the cell lines and tissues examined expressed the alternatively spliced transcript in addition to the transcript coding for the enzymatically functional HPFK-M polypeptide.