We recently showed that phanoside, a gypenoside isolated from the plant Gynostemma pentaphyllum, stimulates insulin secretion from rat pancreatic islets. To study the mechanisms by which phanoside stimulates insulin secretion. Isolated pancreatic islets of normal Wistar (W) rats and spontaneously diabetic Goto-Kakizaki (GK) rats were batch incubated or perifused. At both 3 x 3 and 16 x 7 mM glucose, phanoside stimulated insulin secretion several fold in both W and diabetic GK rat islets. In perifusion of W islets, phanoside (75 and 150 microM) dose dependently increased insulin secretion that returned to basal levels when phanoside was omitted. When W rat islets were incubated at 3 x 3 mM glucose with 150 muM phanoside and 0 x 25 mM diazoxide to keep K-ATP channels open, insulin secretion was similar to that in islets incubated in 150 microM phanoside alone. At 16 x 7 mM glucose, phanoside-stimulated insulin secretion was reduced in the presence of 0 x 25 mM diazoxide (P<0 x 01). In W islets depolarized by 50 mM KCl and with diazoxide, phanoside stimulated insulin release twofold at 3 x 3 mM glucose but did not further increase the release at 16 x 7 mM glucose. When using nimodipine to block L-type Ca2+ channels in B-cells, phanoside-induced insulin secretion was unaffected at 3 x 3 mM glucose but decreased at 16 x 7 mM glucose (P<0 x 01). Pretreatment of islets with pertussis toxin to inhibit exocytotic Ge-protein did not affect insulin response to 150 microM phanoside. Phanoside stimulated insulin secretion from Wand GK rat islets. This effect seems to be exerted distal to K-ATP channels and L-type Ca2+ channels, which is on the exocytotic machinery of the B-cells.