Mutations in carbohydrate-negative mutants of Pseudomonas aeruginosa PAO1 individually deficient in glucose 6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), or pyruvate carboxylase (pyc) were mapped on the chromosome by plasmid R68.45-mediated conjugation and by bacteriophage F116L-mediated transduction. Loci for all three genes were located in the 45- to 55-min region of the chromosome; both zwf-1 and edd-1 were linked by transduction to nalA, whereas pyc-2 was linked by conjugation to argF10. The zwf-1 mutation exhibited cotransduction frequencies of greater than 95% with both edd-1 and the hex-9001 marker, a mutation reported to prevent growth on hexoses. The latter mutation was shown to cause a specific deficiency in 2-keto-3-deoxy-6-phosphogluconate aldolase activity and was redesignated eda-9001. These results demonstrate tight clustering of the gene loci for glucose 6-phosphate dehydrogenase and for both enzymes unique to the Entner-Doudoroff pathway in P. aeruginosa. Our evidence suggests supraoperonic clustering of these and other inducible carbohydrate catabolic genes in the 45- to 55-min region of the chromosome.