Intracellular poly[D-(-)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share approximately 30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (Delta phaZ1, Delta phaZ2, Delta phaZ3, Delta phaZ1 Delta phaZ2, Delta phaZ1 Delta phaZ3, Delta phaZ2 Delta phaZ3, and Delta phaZ1 Delta phaZ2 Delta phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated approximately 80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.