We succeeded in developing a novel method for in vitro poly(3-hydroxybutyrate-co-4-hydroxy-butyrate) [P(3HB-co-4HB)] synthesis with CoA recycling using polyhydroxyalkanoate synthase and an acyl-CoA synthetase. Using this method, the monomer compositions in P(3HB-co-4HB)s could be controlled strictly by the ratios of the monomers in the reaction mixtures.