The pfk gene encoding phosphofructokinase (Pfk) from the anaerobic bacterium Clostridium acetobutylicum ATCC 824 was cloned and sequenced. The gene was identified in a plasmid library by complementation of an E, coli pfk mutant and by the ability to amplify a fragment by PCR using primers based on homologous regions of Pfk from other microorganisms. Nucleotide sequence analysis revealed a coding region for a 319-aa protein homologous to Pfks from other organisms, Enzyme assay and ability to complement the growth defects of E, coli pfk mutants confirmed the expression of the clostridial pfk gene. The pyruvate kinase (pyk) gene was identified adjacent pfk. Such an arrangement for the genes encoding key regulators of glycolytic flux had not yet been described in a strict anaerobe. This gene arrangement has been found in other Gram-positive organisms, but not in Gram-negative organisms.