Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRC(YB4) is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys(151), Asp(306), and His(335)) in the PhaC(YB4) subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRC(YB4) mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRC(YB4)(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRC(YB4) might be elicited by the C151S mutation.