Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia eutropha PHB synthase purified from recombinant E. coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium. Several lines of evidence suggest that the homodimer is the active form of the synthase. The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site. The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified. It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site. To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C-319 mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured. The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis. Two identical thiol groups from C-319 residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions. We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase. These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme.