A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha(Ps-1) and pha(Ps-2)) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The phaPs-2 locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1(Ps-2)-phaCl(Ps-2)-phaZ(Ps-2)-phaC2(Ps-2)phaD(Ps-2). The pha(Ps-1) locus that contains phaCl(Ps-1)-phaZ(Ps-1) appears to have arisen by a duplication event that placed it downstream of a gene (ORF1(Ps-1)), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaCl(Ps-1) and phaCl(Ps-2) were investigated by heterologous expression in Wautersia eutropha PHB(-)4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1(Ps-1) to confer PHA synthesis, however, appeared reduced compared to that of PhaC1(Ps-2), since cells harboring PhaC1(Ps-1) accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1(Ps-2). Primary sequence analysis revealed that PhaC1(Ps-1) had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1(Ps-2)). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1(Ps-1) produces a protein product in the native strain, it is likely that PhaC1(Ps-1) may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.