The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaC_Ca) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp -ketothiolase gene (phaA_Ca). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaA_Ca. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism's PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.