A novel insulin-secreting cell line (BRIN-BD11) was established after electrofusion of RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Wells of cell fusion mixture with insulin output 5-10 times greater than parent RINm5F cells were subcultured with eventual establishment of clones, including BRIN-BD11. Morphological studies established that these cells grow as monolayers with epithelioid characteristics, maintaining stability in tissue culture for > 50 passages. Culture of these cells for 24 h at 5.6-33.3 mmol/l glucose revealed a 1.8- to 2.0-fold increase of insulin output compared with 1.4 mmol/l glucose. Dynamic insulin release was recorded in response to 16.7 mmol/l glucose, resulting in a rapid threefold insulin secretory peak followed by a sustained output slightly above basal. In acute 20-min tests, 4.2-16.7 mmol/l glucose evoked a stepwise two- to three-fold stimulation of insulin release. 3-Isobutyl-1-methylxanthine (1 mmol/l) served to increase basal and glucose-stimulated insulin release, shifting the threshold from 4.4 to 1.1 mmol/l glucose. Stimulation of insulin secretion with 16.7 mmol/l glucose was abolished by mannoheptulose or diazoxide (15 or 0.5 mmol/l). In contrast, glyceraldehyde (10 mmol/l) and 25 mmol/l K+ evoked 1.7- to 9.0-fold insulin responses. L-Alanine (10 mmol/l) evoked a twofold secretory response, which was potentiated 1.4-fold by increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l. Forskolin (25 mumol/l) and phorbol 12-myristate 13-acetate (10 nmol/l) both increased insulin secretion in the presence of L-alanine (1.4- and 1.8-fold, respectively). Western blotting confirmed that BRIN-BD11 cells expressed the GLUT2 glucose transporter. This, coupled with a high glucokinase/hexokinase ratio in the cells, confirms an intact glucose sensing mechanism. High-performance liquid chromatography analysis demonstrated that insulin was the major product secreted under stimulatory conditions. Collectively, these data indicate that the BRIN-BD11 cell line represents an important stable glucose-responsive insulin-secreting beta-cell line for future studies.