The gluconate (gnt) operon of Bacillus subtilis consists of four gnt genes; the second and third genes code for gluconate kinase (gluconokinase, EC 18.104.22.168) and gluconate permease, respectively. A fragment carrying the promoter of this operon (gnt promoter) and the first gene (gntR) was subcloned into a promoter probe vector (pPL603B). Repression of the expression of cat-86 gene, encoded in the vector portion of a constructed plasmid (pgnt21), that is under the control of the gnt promoter was removed by gluconate. The results of deletion analysis and of insertional inactivation of the gntR gene cloned in pgnt21 suggested that the product of the gntR gene, actually synthesized as a 29-kDa protein in vivo, is involved in repression of the gnt promoter. A 4-base-pair insertional mutation within the gntR gene constructed in vitro was introduced into the B. subtilis chromosomal gnt operon by use of linkage of the 4 base pairs to gntK10 in transformation. The introduced mutation gntR1 caused the constitutive expression of the gluconate kinase and gluconate permease genes. S1 nuclease analysis indicated that the mRNA of this operon is synthesized in the gntR1 strain and amounts of mRNA are not changed very much by gluconate, which acts as an inducer in the wild-type gene. These results strongly indicate that the gntR gene codes for a transcriptional negative regulator for the gnt operon.