Mutations causing deficiencies in the inducible, membrane-associated sn-glycerol-3-phosphate dehydrogenase (glpD) and in inducible glucose transport (glcT) were mapped on the Pseudomonas aeruginosa PAO1 chromosome by using the generalized transducing phages F116L and G101. These mutations, in separate catabolic regulatory units, were cotransducible with a previously described cluster of carbohydrate catabolic gene loci (zwf-1 eda-9001 edd-1) that maps at ca. 50 to 53 min on the chromosome. Mutant strain PFB362 (glcT1) did not transport glucose and did not produce a functional, periplasmic, glucose-binding protein that is required for glucose transport. This mutation was cotransducible with zwf-1 (70%), nalA (29%), and phe-2 (19%) but not with glpD1 or leu-10. The glpD1 mutation in strain PRP408 was cotransducible with zwf-1 (5%), eda-9001 (4%), and edd-1 (1%) and also with ami-151 (17%) and phe-2 (33%). These results expand the number of known carbohydrate catabolism genes that are clustered in the 50- to 55-min region of the PAO1 chromosome and allow us to propose the following relative gene order: ami-151 glpD1 phe-2 nalA zwf-1 eda-9001 edd-1 glcT1 leu-10. Three independently obtained nal determinants for high-level resistance to nalidixic acid, which were employed in these studies, exhibited similar cotransduction frequencies with several flanking marker mutations.