Various hydroxyacyl coenzyme A (COA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-C-14]D-(-)-hydroxybutyric acid, [1-C-14]D-lactic acid, [1-C-14]L-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, D-(-)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5’-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-C-14]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-C-14]lactyl-CoA was used as a substrate at a rate that was only less than 10(-4) of the rate than with [3-C-14]D-(-)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-C-14]d-lactyl-CoA as substrate at a relatively high rate.