From genomic libraries, the polyhydroxyalkanoate gene locus of Pseudomonas aeruginosa PAO1 was cloned and characterised at the molecular level. Two genes coding for polyhydroxyalkanoate synthases, phaC1Pa and phaC2Pa, a polyhydroxyalkanoate depolymerase gene, phaDPa, and four adjacent open reading frames (ORF1, ORF2, ORF3 and ORF4) were identified from the nucleotide sequence. Two transcriptional start sites, which were preceded by sequences resembling the Escherichia coli consensus sequences for σ54 and σ70 promoters, were identified experimentally upstream of phaC1Pa, which was shown by Northern blot analysis to constitute an operon together with phaDPa. A third putative promoter resembling the E. coli consensus sequence for σ70-dependent promoters was proposed upstream of phaC2Pa, which is in a bicistronic operon with ORF3. Investigations of rpoN-negative mutants of related strains revealed that polyhydroxyalkanoate accumulation from gluconate required an intact rpoN locus in P. aeruginosa. Complementation experiments revealed multiple evidence that either polyhydroxyalkanoate synthase is involved in polyhydroylkanoate accumulation from gluconate as well as from octanoate. The P. aeruginosa PAO1 polyhydroxyalkanoate gene locus was expressed in the polyhydroxyalkanoate-negative mutant Alcaligenes eutrophus PHB−4 and in the poly(3-hydroxybutyrate)-accumulating strain P. oleovorans DSM1045. It conferred on the latter the ability to synthesize and accumulate polyhydroxyalkanoates consisting of medium-chain-length 3-hydroxyalkanoic acids from unrelated substrates in addition to poly(3-hydroxybutyrate). The sequence of the putative translational product of ORF1 was similar to those of the leukotoxin repressor of Pasteurella haemolytica and to the ORF9 product of Azotobacter vinelandii, and that of ORF4 was similar to the algP product of P. aeruginosa and to eukaryotic histone H1 proteins. The proteins of ORF2 and ORF3 appear to be previously unidentified.