The gene (Bs-pfk) for phosphofructokinase (PFK) from Bacillus stearothermophilus has been cloned and sequenced. The deduced amino acid sequence is nearly identical to the sequence which was previously determined by peptide analysis. The elevated G + C content of Bs-pfk relative to the homologous Ec-pfkA from Escherichia coli is consistent with previous observations concerning genes from thermophilic prokaryotes. A significant degree of homology exists when the deduced amino acid sequence of B. stearothermophilus PFK is compared with the corrected sequences of rabbit muscle PFK or E. coli PFK-1. The cloning and sequencing of Bs-pfk completes the first step toward using site-specific mutagenesis to investigate the structure-function relationships for this allosteric enzyme.