Two new 3-hydroxyalkanoates, 3-hydroxy-3-cyclopropylpropionate (3CyP3HP) and 3-hydroxy-4-chlorobutyrate (4C13HB), were prepared, and their coenzyme A (CoA) thioester derivatives were polymerized in aqueous solutions with the polymerase enzyme of a bacterium which can produce poly-3-hydroxyalkanoates (PHAs) as intracellular, reserve polymers. The polymerase used for the in vitro polymerization reactions was that of E. shaposhnikovii (EsPHAS), which was produced by a recombinant strain of E. coli harboring the appropriate gene. The polymerase of R. eutropha (RePHAS) was also evaluated for the in vitro polymerization of the two monomers, but it was inactive with the CoA thioester of 3CyP3HP. The propagation rates of the two monomers with the EsPHAS polymerase were 1.2 and 6.7 mol of monomer reacted per mole of enzyme catalyst per second of reaction time for 3CyP3HPCoA and 4C13HBCoA, respectively, and the number-average molecular weights of the PHAs obtained were 371 000 and 189 000, respectively. Both polymers were crystalline, and their crystallinity and NMR spectra indicated that both contained only one chiral center (100% isotactic) even though both of the monomers were racemic mixtures.