Morphological analysis of hormone content and functional assessment of hormone secretion were conducted in βTC-6 cells, an insulin-secreting cell line derived from transgenic mice expressing the large T-antigen of simian virus 40 (SV40) in pancreatic β-cells. We observed by immunohistochemistry and confocal microscopy that βTC-6 cells contain abundant insulin and small amounts of glucagon and somatostatin (SRIF). Glucagon usually co-localized with insulin, whereas cells containing SRIF did not contain insulin or glucagon. Static incubation and perifusion experiments demonstrated that βTC-6 cells at passage 30-45 secrete insulin in response to glucose. In static incubations, mammal stimulation was achieved for glucose concentrations >2.8 mmol/l glucose, and the half-maximal effect was observed at 0.5 mmol/l. Maximal stimulation was four times greater than HIT-T15 cells at passage 72-81, although HIT cells had a greater response over their basal levels. The magnitude of the insulin response to glucose hi perifusion was 1,734 ± 384 pmol · l-1 · min and was 4.6-fold greater hi the presence of 3-isobutyl-1-methylxanthine. Low amounts of glucagon were released in response to amino acids. Epinephrine (EPI), and to a lesser extent SRIF, inhibited phasic glucose-induced insulin secretion. A major portion of these inhibitory effects was mediated by pertussis toxin-sensitive substrates. Immunoblots detected the presence of the G-proteins Giα2, Giα3, and Goα2. These results indicate that βTC-6 cells are a glucose-responsive cell line in which insulin exocytosis is physiologically regulated by EPI and SRIF through Gi/Go-mediated mechanisms.