Aims: To investigate the regulation of de novo pyrimidine biosynthesis in the polyhydroxyalkanoate-producing bacterium Pseudomonas oleovorans at the level of enzyme synthesis and at the level of aspartate trans carbamoylase activity. Methods and Results: The effect of pyrimidine supplementation on the pyrimidine biosynthetic pathway enzyme activities was analysed relative to carbon source. Two uracil auxotrophs of P. oleovorans were isolated that were deficient for aspartate transcarbamoylase or dihydroorotase activity. Pyrimidine limitation of these auxotrophs increased the de novo pathway activities to varying degrees depending on the pathway mutation and the carbon source utilized. At the level of aspartate transcarbamoylase activity, pyrophosphate and uridine ribonucleotides were found to be strongly inhibitory of the Ps. oleovorans enzyme. Conclusions: Pyrimidine biosynthesis is regulated in Ps. oleovorans. Taxonomically, the regulation of the pyrimidine biosynthetic pathway appeared dissimilar from previously studied Pseudomonas species. Significance and Impact of the Study: New insights regarding the regulation of nucleic acid metabolism are provided that could prove significant during the genetic manipulation of Ps. oleavorans to increase the synthesis of polyhydroxyalkanoates.