The enzymes involved in gluconate utilization in Bacillus subtilis seemed to be gluconate permease and gluconate kinase. Several mutants unable to grow on gluconate were isolated. The mutations they harboured (gnt) were clustered between iol-6 and fdp-74 on the B. subtilis chromosome (a tentative map order of gnt-10, gnt-4, gnt-26, gnt-23 and gnt-9 was obtained). The gnt-10 mutation seemed to be located within the structural gene of the kinase, and the gnt-23 and gnt-26 mutations seemed to be within that of the permease. An EcoRI fragment (4.5 MDal) containing an intact gluconate (gnt) operon consisting of these two structural genes was cloned in phage phi 105 by prophage transformation and was mapped physically. The physical location of the mutations coincided with their order on the genetic map. The HindIII-A fragment (2.4 MDal), which corrects all the gnt mutations, was subcloned in plasmid pC194. The fragment contained the structural genes for the gluconate permease and kinase, but not the regulatory region of the gluconate operon.