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Poly-β-hydroxyalkanoate (PHA) from crotonate-grown cultures of Syntrophomonas wolfeicontained only the d-isomer of β-hydroxybutyrate. The PHA from cultures grown with trans-2-pentenoate or one of several hexenoates as the substrate also contained small amounts (5%) of β-hydroxypentanoate or β-hydroxyhexanoate, respectively. Thus, some PHA was synthesized without cleavage of the carbon skeleton of the substrate, but the predominant route for PHA synthesis was by the condensation and subsequent reduction of acetyl-coenzyme A (CoA). The ratio of the β-hydroxypentanoate to the β-hydroxybutyrate in PHA in pentenoate-grown cultures increased immediately after inoculation and then decreased as the amount of the β-hydroxybutyrate in PHA increased. The amount of β-hydroxypentanoate in the PHA did not markedly change throughout the remainder of growth. These data indicated that the unbroken carbon-chain was used for polymer production only in the early stages of growth and, later, polymer synthesis occurred by the condensation and reduction of acetyl-CoA molecules.