The enzyme, phosphotransacetylase (Pta), catalyzes the conversion of acetyl coenzyme A to acetyl phosphate. The putative pta gene of Bacillus subtilis, which had been sequenced as part of the Genome Project, was cloned and overexpressed in Escherichia coli. We confirmed that the gene encodes Pta by measuring the enzymatic activity of the purified protein. Insertional mutagenesis of the pta gene resulted in complete loss of the Pta activity, indicating that B. subtilis contains only one kind of pta gene. Expression of a pta-lacZ fusion was induced in the presence of excess glucose in the growth medium, and the intact ccpA gene was required for this activation. The transcriptional start site of the pta gene was located at 37 nucleotides upstream of the pta start codon, and a cre (catabolite responsive element) sequence, a cis-acting element that is responsible for the catabolite repression of a number of carbon utilization genes in B. subtilis, was identified upstream of the tentative promoter site. Experiments involving oligonucleotide-directed mutagenesis showed that the cre sequence is involved in glucose-mediated transcriptional activation.