Previous investigations on the role of the polymerase in the synthesis of poly-3-hydroxybutyrate (PHB) are reviewed, and the results from earlier in vitro studies on the activity and selectivity of the polymerase of Alcaligenes eutrophus are discussed. In the present study the effect of glycerol on stabilizing the polymerase after purification and on eliminating the lag phase in in vitro polymerization reactions of 3-hydroxybutyl CoA (HBCoA), and 3-hydroxyvaleryl CoA (HVCoA) are described. K(M) values were determined for the activity of the polymerase with both HBCoA and HVCoA, and the rates of propagation for both monomers were estimated. With a racemic mixture of HBCoA, the enzyme polymerized only the [R] monomer.