The amino acid sequence of an intracellular poly[D(-)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ1) from Ralstonia eutropha H16 was compared with the sequences of various proteins using the BLAST search. It showed a number of matches including with intracellular PHB depolymerases, conserved hypothetical proteins, and PHB synthases. From an alignment of these proteins, we constructed nine mutants: C87A, S118A, H120Q, C183A, C183S, D355A, D356A, C370A, and H388Q. The C183A, D355A, and H388Q mutants lost their activities, but C183S and the other mutants did not. C183, D355, and H388 in PhaZ1 were positioned similarly to the amino acids of the catalytic triad of PHB synthase. These results indicated that C183, D355, and H388 make up the catalytic triad of PhaZ1.