To determine if endogenous gamma-aminobutyric acid (GABA) is secreted by a pancreatic beta-cell-derived cell line and to determine the effects of glucose on GABA release, beta TC6 cultures were incubated in the presence of 1 or 10 mmol/l glucose for 12 h and then subjected to a 2-h secretion test in Krebs-Ringer buffer containing 1 or 10 mmol/l glucose. beta TC6-conditioned medium was collected at 15, 30, 60, and 120 min after glucose stimulation for GABA analysis by high pressure liquid chromatography-electrochemical detection. After 30 min, medium GABA concentrations were significantly higher (p < 0.05) in cultures that were exposed to high glucose during both the 12-h incubation period and the 2-h secretion test than in the remaining three glucose combinations. To address possible roles of beta-cell-derived GABA, the effect of GABA on glucagon secretion from pancreatic alpha TC6 cells was tested at concentrations released from beta TC6 cells. Inhibition of glucagon secretion by alpha TC6 cells was observed in the presence of GABA at concentrations equivalent to concentrations secreted by beta TC6 cells. The inhibitory effects of GABA on glucagon secretion by alpha TC6 cells were blocked by the GABAA receptor antagonist bicuculline and were dissociated from the inhibitory effects of glucose. Together, these results provide the first documentation that endogenous GABA is released from a highly differentiated beta-cell line and that glucose and GABA independently attenuate glucagon secretion by a pancreatic alpha-cell line.