The generation and characterization of Pseudomonas putida KT2442 mutants affected in poly-3-hydroxyalkanoate (PHA) synthesis are reported. The mutants from P. putida KT2442 carrying several copies of the PHA-polymerase-encoding gene (phaC) were isolated via N-methyl-N＇-nitro-N-nitrosoguanidine chemical mutagenesis and contained mutation(s) on genes that are involved in PHA accumulation other than the phaC genes. No PHA-free mutants were obtained, suggesting that there must be various routes for the synthesis of PHA polymerase precursors. One of the isolated mutants (GPp120) accumulated more PHA than the parental strain, and there was virtually no down-regulation of PHA formation by growth in nonlimiting amounts of nitrogen, which normally block or reduce formation of PHA. Compared to the parental strain. GPp120 exhibited significant changes in physiology and morphology when grown in minimal medium : the growth rate was reduced more than twofold and cells formed filaments. The other four groups of isolated mutants, with P, putida strains GPp121 to GPp124 as characteristic type strains, exhibited morphological characteristics similar to those of the parental strain. However, they showed reduced PHA production compared to the parental PHA(+) strain, and especially GPp121 and GPp122 showed PHA formation tightly controlled by nutrient conditions. All of these mutants provide starting points for genetically dissecting the biosynthesis and regulation of PHA precursors.