Thiolase of Clostridium acetobutylicum is an important enzyme involved in both, acid and solvent fermentation. Two thiolase genes (thlA and thIB) have been cloned and sequenced from Clostridium acetobutylicum DSM 792, showing high homology to each other and to thiolases of PHA-synthesizing bacteria. The thlA gene is identical to the gene already cloned and sequenced from strain ATCC 824 (Stim-Herndon et al., 1995, Gene 154: 81-85). Using primer extension and S1 nuclease analysis a transcriptional start site was identified 102 bp upstream of the thlA start codon. This site was preceded by a region that exhibits high similarity to the sigma70 consensus promoter sequences of Gram-positive and -negative bacteria. Regulation of thlA and thlB was studied at the transcriptional level to elucidate the specific function of each gene. Non-radioactive primer extension analysis using fluorescein-labelled oligonucleotides and Northern blot analysis revealed high levels of thlA transcripts in acid- and solvent-producing cells. During an induced shift of a continuous culture from acid to solvent formation, the transcript level transiently decreased to a minimum, 3 to 7 h after induction. The thlA transcript length is about 1.4 kb, indicating a monocistronic organisation, whereas genetic organization and reverse transcription (RT)-PCR analysis indicated that thlB forms an operon with two other adjacent genes, thlR and thlC. Transcription and regulation of the thlB operon was studied using RTPCR and showed a very low expression in acid- and solvent-producing cells. Heterologously expressed clostridial ThlB showed high thiolase activity in Escherichia coli. The N-terminal part of ThlR possesses a potential helix-turn-helix motif and shows significant homology to regulatory proteins belonging to the TetR/AcrR family of transcriptional regulators. ThlR possibly acts as a transcriptional repressor of thlB operon expression. The data provide strong evidence that ThlA is involved in the metabolism of both acid and solvent formation, whereas the physiological function of ThlB has yet to be elucidated.