Nicotine elicited membrane depolarization, elevation of intracellular calcium, rubidium efflux, and release of insulin from mouse ␤-TC6 insulinoma cells. Such responses were blocked by the nicotinic antagonist mecamylamine but not by the muscarinic antagonist atropine. Neither the selective ␣ 4 ␤ 2 antagonist dihydro-␤-erythroidine nor the selective ␣ 7 antagonist methyllycaconitine significantly blocked the nicotine-elicited depolarization or the calcium response. The elevation of intracellular calcium did not occur in calcium-free media, indicating that the increase in intracellular calcium was due to the influx of calcium. The rank order of potency for nicotinic agonists was as follows: epibatidine Ͼ nicotine ϭ 3-(azetidinylmethoxy)pyridine (A-85380), cytisine, dimethylphenylpiperazinium (DMPP). Cytisine and DMPP seemed to be partial agonists. The density of nicotinic receptors measured by [ 3 H]epibatidine binding was 7-fold higher in membranes from ␤-TC6 cells than in rat brain membranes. No binding of 125 I-A-85380 was detected, indicating the absence of ␤2-containing receptors. Reverse transcription-polymerase chain reaction analyses indicated the presence of mRNA for ␣3 and ␣4 subunits and ␤2 and ␤4 subunits in ␤-TC6 cells. The binding and functional data suggest that the major nicotinic receptor is composed of ␣3 and ␤4 subunits. The ␤-TC6 cells thus provide a model system for pharmacological study of such nicotinic receptors.