The whole polyhydroxyalkanoate (PHA) synthesis gene locus of Pseudomonas stutzeri strain 1317 containing PHA synthase genes phaC1 Ps, phaC2 Ps and PHA depolymerase gene phaZ Ps was cloned using a PCR cloning strategy. The sequence analysis results of the phaC1 Ps, phaC2 Ps and phaZ Ps showed high homology to the corresponding pha loci of the known Pseudomonas strains, respectively. PhaC1 Ps and PhaC2 Ps were functionally expressed in recombinant Escherichia coli strains and their substrate specificity was compared. The results demonstrated that PhaC1 Ps and PhaC2 Ps from P. stutzeri 1317 had different substrate specificities when expressed in E. coli. In details, PhaC2 Ps could incorporate both short-chain-length 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates (mcl 3HA) into PHA, while PhaC1 Ps only favored mcl 3HA for polymerization.