Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assess the PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulin release (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 mM) enhanced PIR ~ 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by ~ 40-50%. Both effects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cch also evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronic TMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. In contrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-induced insulin secretion.