Paracoccus denitrificans PD1222 accumulates short-length polyhydroxyalkanoates, poly(3-hydroxybutyrate), under nitrogen-deficient conditions. Polyhydroxybutyrate metabolism requires the 3-ketoacyl-CoA thiolase PhaA, the acetoacetyl-CoA dehydrogenase/reductase PhaB and the synthase PhaC for polymerization. Additionally, P. denitrificans PD1222 grows aerobically with nitrate as sole nitrogen source. Nitrate assimilation is controlled negatively by ammonium through the two-component NtrBC system. NtrB is a sensor kinase that autophosphorylates a histidine residue under low-nitrogen concentrations and, in turn, transfers a phosphoryl group to an aspartate residue of the response regulator NtrC protein, which acts as a transcriptional activator of the P. denitrificans PD1222 nasABGHC genes. The P. denitrificans PD1222 NtrB mutant was unable to use nitrate efficiently as nitrogen source when compared to the wild-type strain, and it also overproduced poly(3-hydroxybutyrate). Acetyl-CoA concentration in the P. denitrificans PD1222 NtrB mutant strain was higher than in the wild-type strain. The expression of the phaC gene was also increased in the NtrB mutant when compared to the wild-type strain. These results suggest that accumulation of poly(3-hydroxybutyrate) in the NtrB mutant strain of PD1222 responds to the high levels of acetyl-CoA that accumulate in the cytoplasm as consequence of its inability to efficiently use nitrate as nitrogen source.