Several recombinant Escherichia coli strains harboring the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes were used to produce poly(3-hydroxybutyrate), PHB, from xylose. By flask culture of TG1 (pSYL107) in a defined medium containing 20 g/l xylose, PHB concentration of 1.7 g/l was obtained. Supplementation of a small amount of cotton seed hydrolysate or soybean hydrolysate could enhance PHB production by more than two fold. The PHB concentration, PHB content, and PHB yield on xylose obtained by supplementing soybean hydrolysate were 4.4 g/l, 73.9%, and 0.226 g PHB/g xylose, respectively.