The aim of this study was to evaluate the hypothesis that Γ-aminobutyric acid (Gaba) synthesis in situ is regulated by Glu availability. The Gly decarboxylase (EC 126.96.36.199) inhibitor, aminoacetonitrile (AAN), was investigated as a means of preventing the recycling of photorespiratory ammonia, thereby causing the accumulation of Glu. In vitro measurements of crude tobacco (Nicotiana tabacum L. cv. Samsun NN) leaf extracts demonstrated that AAN effectively inhibited Gaba:pyruvate transaminase (EC 188.8.131.52) activity, but not Glu decarboxylase (EC 184.108.40.206), Glu:oxaloacetate transaminase (EC 220.127.116.11) or Glu:pyruvate transaminase (EC 18.104.22.168) activities. Gly decarboxylase activity was inhibited preferentially over Gaba transaminase, and secondary effects that can lead to an inhibition of photosynthesis were minimized by the use of a short-time course of only 90 min to investigate the impact of 25 mM AAN on the steady state metabolism of [U-14C]Glu by young transpiring shoots of tobacco seedlings. With time, AAN treatment caused the accumulation of Gly/Asn, but not Gaba. Furthermore, AAN increased the C-radioactivity in Glu, Pro and Krebs cycle organic acids, and reduced the C-radioactivity in Gln and Gaba. The data provide evidence for increased flux of carbon through the Gaba shunt when the availability of Glu as a substrate for Glu decarboxylase was enhanced.