The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of the polyhydroxyalkanoate produced from glucose byRhodococcus ruberwas investigated using nuclear magnetic resonance analysis. Spectra obtained for polymer produced from [6-13C]-, [2-13C]-, and [1-13C]glucose were compatible with carboxylation of pyruvate followed by TCA cycle reactions to yield succinate, which is a precursor of propionyl-CoA in the biosynthesis of 3HV in this organism. Carboxylation of pyruvate may occur directly or involve methylmalonyl-CoA transcarboxylase but the latter process is more probable as it provides a route for recycling oxaloacetate, which is essential for the pathway to operate. The observed incorporation of label from NaH13CO3into C-3 of 3HV monomers cannot occur via forward reactions of the TCA cycle but may be explained by direct carboxylation of pyruvate, followed by reverse reactions of the TCA cycle to yield succinyl-CoA. This minor route accounts for at least 12% of the carbon flux in the synthesis of 3HV monomer units. Inhibitors of key enzymes of possible pathways for polyhydroxyalkanoate biosynthesis inR.ruberwere tested. Addition of monofluoroacetate, an inhibitor of aconitase, to cultures decreased production of 3HV, supporting the proposed major route for 3HV synthesis.Key words:Rhodococcus ruber, polyhydroxyalkanoate, PHA, biosynthesis.