Cx36 is the predominant connexin isoform expressed by pancreatic beta-cells. However, little is known about the role of this protein in the functioning of insulin-secreting cells. To address this question, we searched for a cell line expressing Cx36 and having glucose-induced insulin secretion comparable to that of primary beta-cells. By evaluating Cx36 expression in MIN6, betaTC3, RIN2A, INS1, and HIT cell lines, which differ in their sensitivity to glucose, we found that wild-type MIN6 cells fit these requirements. Therefore, we stably transfected MIN6 cells with a cDNA coding for a Cx36 antisense sequence to study the role of Cx36 in these cells. Independent clones of MIN6 cells were obtained that had a markedly reduced Cx36 expression. Loss of Cx36 decreased functional gap junctional conductance in these clones. This alteration impaired the synchronization of glucose-induced [Ca(2+)](i) oscillations and insulin secretion in response to glucose, to secretagogues that increase [cAMP](i), and to depolarizing conditions. These data provide the first evidence that Cx36-made channels 1) mediate functional coupling in MIN6 cells, 2) provide for synchronous [Ca(2+)](i) oscillations, and 3) are necessary for proper insulin secretion in response to metabolizable and nonmetabolizable secretagogues.