The enzymes phosphotransacetylase (PTA) and acetate kinase (AK) catalyze the conversion of acetyl coenzyme A to acetate in the fermentation of Clostridium acetobutylicum. The acetate-producing step is an important element in the acidogenic fermentation stage and generates ATP for clostridial cell growth. The genes pta and ack, encoding PTA and AK, respectively, were cloned and sequenced. Enzyme activity assays were performed on cell extracts from Escherichia coli and C. acetobutylicum harboring the subclone, and both AK and PTA activities were shown to be elevated. DNA sequence analysis showed that the pta and ack genes are adjacent in the clostridial chromosome, with pta upstream. The pta gene encodes a protein of 333 amino acid residues with a calculated molecular mass of 36.2 kDa, and ack encodes a polypeptide of 401 residues with a molecular mass of 44.3 kDa. Primer extension analysis identified a single transcriptional start site located 70 bp upstream of the start codon for the pta gene, suggesting an operon arrangement for these tandem genes. The results from overexpression of ack and pta in C. acetobutylicum showed that the final ratios of acetate to other major products were higher and that there was a greater proportion of two- versus four-carbon-derived products.