Earlier studies suggest that the accumulation of non-esterified arachidonic acid (AA) in islets following stimulation with glucose participates in the glucose-induced secretion of insulin. A possible role for AA might include the facilitation of Ca2+ influx into islet β-cells. Recently, we demonstrated that AA induces Ca2+ influx into purified rat pancreatic islet β-cells, prepared by fluorescence-activated cell sorting (FACS). This effect was abolished in the presence of the Ca2+-chelator EGTA, but was only partially reduced by the dihydropyridine (DHP) L-type Ca2+-channel blocker, nifedipine. This raised the possibility that DHP-insensitive Ca2+ entry mechanisms may exist in pancreatic β-cells, in addition to the known DHP-sensitive L-type Ca2+ channels. Here we report that ω-conotoxin (CTX), which blocks ω-type Ca2+-channels, inhibits AA-induced Ca2+ influx by a magnitude similar to that of nifedipine and that the combination of ω-CTX and nifedipine results in a nearly additive decrement in AA-induced increases in β-cell cytosolic [Ca2+]. We further demonstrate that bovine serum albumin, which complexes free AA and prevents AA-induced increases in cytosolic [Ca2+], also inhibits the glucose-induced increase in β-cell [Ca2+]. These results suggest that rat pancreatic FACS-purified islet β-cells express ω-type (DHP-insensitive) Ca2+-channels, in addition to DHP-sensitive Ca2+-channels. They further suggest that the glucose-induced accumulation of non-esterified AA in the membranes of β-cells serves to amplify glucose-mediated Ca2+ influx into the β-cells.