Gamma amino butyrate (GABA) is the major inhibitory neurotransmitter in the nervous system. The compound is also released by the insulin-producing pancreatic beta cells providing them with a potential paracrine regulator. Since glucose was found to inhibit GABA release we investigated whether extracellular GABA can serve as a marker for glucose-induced mitochondrial activity, and thus for the functional state of beta cells. GABA release by rat and human beta cells was shown to reflect net GABA production varying with the functional state of the cells. Net GABA production is the result of GABA formation through glutamate decarboxylase (GAD) and GABA catabolism involving a GABA-transferase (GABA-T) mediated shunt to the TCA-cycle. GABA-T exhibits Km values for GABA (1.25mM) and for alpha-ketoglutarate (-KG -0.49mM) that are, respectively, similar and lower than those in brain. The GABA-T inhibitor gamma-vinyl GABA was used to assess the relative contribution of GABA formation and catabolism to net production and release. The nutrient status of the beta cells was found to regulate both processes. Glutamine dose-dependently increased GAD-mediated formation of GABA whereas glucose metabolism shunts part of this GABA to mitochondrial catabolism, involving -KG-induced activation of GABA-T. In absence of extracellular glutamine, glucose also contributed to GABA formation through aminotransferase generation of glutamate from -KG; this stimulatory effect only increased GABA release when GABA-T activity was suppressed. It is concluded that the GABA release from beta cells is regulated by the cellular nutrients glutamine and glucose. Glucose inhibits glutamine-driven GABA formation and release through increasing GABA-T shunt activity by its cellular metabolism. Our data indicate that GABA release by beta cells can be used to monitor their metabolic responsiveness to glucose, irrespective of their insulin secretory activity.