A number of Escherichia coli strains including K12, B, W, XL1-Blue, DH5 alpha, HB101, JM109, and C600 were transformed with the stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid biosynthesis genes, and were subsequently compared for their ability to synthesize and accumulate poly(3-hydroxybutyric acid) (PHB). The rate of PHB synthesis, the extent of PHB accumulation, and PHB yield from glucose varied considerably from one strain to another. Strains XL1-Blue and B harboring pSYL105 synthesized PHB at the highest rate to a final concentration of ca. 7 g/L in complex medium containing 20 g glucose/L. With an aim to reduce the cost of the medium, the effect on PHB accumulation of supplementing a defined medium with complex nitrogen sources was examined. A PHB concentration of 81 g/L could be obtained in 41 h from a pH-stat fed-batch culture of XL1-Blue(pSYL105) in a semidefined medium. When the availability of acetyl-CoA was increased by supplementing the medium with complex nitrogen sources, amino acids, or oleic acid, PHB synthesis by recombinant E. coli was enhanced.