The structure and regulation of the gab gene cluster, involved in gamma-aminobutyric acid (GABA) shunt, were studied by characterizing gabT and gabD genes cloned from Bacillus thuringiensis. Deletions of the gabT and gabD genes in B. thuringiensis strain HD-73 did not affect the growth of mutant strains in rich culture media, but the growth of a gabT deletion mutant strain was reduced in basic media (containing 0.2% GABA). Genome analysis indicates that the structure of the gab gene cluster in B. thuringiensis HD-73 is different from that in Escherichia coli and Bacillus subtilis but is common in strains of the Bacillus cereus group. This suggests that the gene cluster involved in GABA shunt is specific to the B. cereus group. Based on reverse transcription-PCR and transcriptional fusion analysis, we confirmed that the gabT and gabD genes belong to different transcriptional units, while the gabD and gabR genes form an operon. We also demonstrated that the gabR gene plays a positive regulatory role in gabD and gabT expression. The GabR protein may be a sigma(54)-dependent transcriptional activator, according to a conserved domain search in the NCBI database, and it is highly conserved in the B. cereus group. The -24/-12 consensus sequence of a promoter upstream from gabT suggests that the promoter can be recognized by a sigma(54) factor. Further analysis of the genetic complementation studies also suggests that the expression of the gabT gene is controlled by a sigma(54) factor. Thus, the expression of the gab cluster is regulated by a sigma(54) factor by way of the transcription activator GabR.