This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P. mendocina pha gene locus, encoding two PHA synthase genes [phaC1(Pm) and phaC2(Pm) flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1(Pm). (1,677 bp), phaZ (1,034 bp), and phaC2(Pm) (1,680 bp) were determined. The amino acid sequences deduced from phaC1(Pm) and phaC2(Pm) showed highest similarities to the corresponding PHA synthases from other pseudo-monads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants R putida GPp104 and Ralstonia eutropha PHB-4. In P.putida GPp104, phaC1(Pm) and phaC2(Pm) mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6-C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB-4, either PHA synthase gene also led to the incorporation of 3-hydroxybuty rate (31413) into PHA. Recombinant strains of R. eutropha PHB-4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC I and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in R mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.