In Clostridium acetobutylicum the three enzymes thiolase, CoA-transferase, and acetoacetate decarboxylase serve to catalyze the formation of acetone from acetyl-CoA. This process is unique to butyric-acid clostridia and has long-term industrial significance. These three enzymes are known to he induced at the onset of solvent production. Both the acetoacetate decarboxylase and CoA-transferase activities are often spontaneously lost in degenerative mutants. To further investigate the regulatory mechanisms governing the expression of these enzymes, the genes encoding each of the three enzymes were cloned and expressed in Escherichia coli. The CoA-transferase is an α\sb2,β\sb2 heterotetramer of 23 and 26 kDa subunits. Cloning revealed the enzyme is encoded by two genes (actA and actB). Genes for both subunits of the CoA-transferase were located in an operon arrangement. DNA sequence analysis suggests that these genes are co-transcribed as part of a larger operon from an upstream promoter. The adc gene, encoding the acetoacetate decarboxylase, was cloned and physically mapped downstream from the CoA-transferase genes. However, it is not part of the actAB operon, being oriented in the opposite direction. DNA sequencing analysis demonstrates transcription of the adc as a monocistronic operon. The 65 bp intergenic region between the C-termini of the adc and actB genes is characterized by a novel stem-loop structure which appears capable of rho-independent transcription termination function in both directions in C. acetobutylticum. The cloned adc gene was successfully reintroduced into a C. acetobutylicum adc\sp− mutant, resulting in wild-type regulation and expression of the gene. The gene encoding the thiolase (thl) was also cloned, although it is not located near the other acetone production genes. The cloned thiolase is transcribed from its own promoter. No other thiolase genes were identified. A profound bias for adenine or thymidine in the third ("wobble") position of each codon was demonstrated in all four genes sequenced. Despite preferential usage of codons rarely used in E. coli, the transcription of the genes, assembly of subunits, and high levels of enzyme activity indicate the genes were well expressed in E. coli.