Phosphoglucoisomerase (pgi) mutations in a number of independently isolated mannitol-negative mutants of Pseudomonas aeruginosa PAO1 were mapped on the chromosome by plasmid FP5-mediated conjugation and by cotransduction with the generalized transducing phages G101 and F116L. Mutant allele pgi-9001 exhibited linkage to ilvB, C-9059 (46-85%), car-9003 (93-100%), and pur-9047 (70%), but not with met-9011, in FP5-mediated conjugational crosses. All known pgi mutations and several previously uncharacterized mannitol-negative mutations exhibited transductional linkage to two independent car mutations at frequencies ranging from 13% to 42% and 53% to 99%, in transductional crosses mediated by phages G101 and F116L respectively. These pgi and mannitol-negative mutations also were cotransducible at very low frequencies (<1%) with two independent ilv mutations. Cotransduction of the car and ilv loci could not be detected. These data suggest the location of pgi within the first minute of the P. aeruginosa chromosome closely linked to the car marker and probably between the ilv and car loci. All of the mannitol-negative mutations that exhibited linkage to the car and ilv loci were characterized as pgi mutations by enzyme assays. A phenotypically similar, mannitol-negative mutant was shown to contain a mutation in glucose-6-phosphate dehydrogenase (zwf-9012) that maps to a different region on the chromosome.