The ptsP, ptsO, and ptsN genes encode Enzyme I(Ntr), NPr, and enzyme IIA(Ntr) (IIA(Ntr)) proteins of the nitrogen-related phosphotransferase system. These proteins participate in a phosphoryl transfer chain in several bacteria, where IIA(Ntr) appears to be the terminal phosphoryl acceptor. Inactivation of the ptsP gene in Azotobacter vinelandii was previously shown to reduce poly-beta-hydroxybutyrate (PHB) production. Therefore, the question of a role of the ptsO and ptsN gene products in PHB synthesis was raised. In this work we constructed strains carrying mutations in the ptsO and ptsN genes and tested their effects on PHB accumulation. In the ptsO mutant, PHB accumulation diminished as in the ptsP mutant, while the ptsN mutant accumulated more PHB than the wild-type strain. The negative effects of the ptsP and ptsO mutations on PHB accumulation was suppressed by the ptsN mutation, and a H68A mutation in the phosphorylatable site of IIA(Ntr), impaired PHB accumulation similar to the ptsP mutation. The ptsP and ptsO mutations negatively affected transcription of the phbBAC biosynthetic operon and of the phbR gene coding for a transcriptional activator of phbBAC, whereas the ptsN mutation increased expression of this operon. Taken together our data provide genetic evidence suggesting that the non-phosphorylated form of IIA(Ntr) is involved in negative regulation of phbR and phbBAC expression in A. vinelandii.