The mutation of Clostridium acetobutylicum mutant AA2, defective in the formation of acetone and butanol, was shown to be caused by a single insertion of Tn916 close to the structural gene thrA, encoding the tRNA(Thr)ACG. The DNA region containing the thrA gene was cloned and sequenced. Start and end points of the transcript were determined by primer extension and S1-mapping analysis. The results obtained were identical to predictions derived from the DNA sequence by various RNA-analysing computer programs. The rarely used ACG codon seems to be confined to genes expressed at the end of the exponential growth phase or involved in uptake or turnover of minor C or N substrates. Evolutionary aspects of this codon selection and a possible translational regulation mechanism are discussed.