The nucleotide sequence of a 2.86-kb cDNA clone containing the complete human muscle phosphofructokinase (PFK) protein-coding region was determined. It comprises 76 bp of 5'-untranslated sequence, 2340 bp encoding human muscle PFK polypeptide, and 399 bp of 3'-untranslated sequence plus a poly(A) tract. A retroviral vector was utilized to express the product of this coding sequence in mouse fibroblasts. The PFK-coding cDNA was shown to code for an enzymatically active polypeptide by immunoprecipitation analysis and DEAE-Sephadex A-25 chromatography.