In pancreatic islets the activation of phospholipase C (PLC) by the muscarinic receptor agonist carbamyolcholine (carbachol) results in the hydrolysis of both phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) and phosphatidylinositol (PtdIns). Here we tested the hypothesis that PtdIns hydrolysis is mediated by PLC␥1, which is known to be regulated by activation of tyrosine kinases and PtdIns 3-kinase. PtdIns breakdown was more sensitive than that of PtdInsP 2 to the tyrosine kinase inhibitor, genistein. Conversely, the tyrosine phosphatase inhibitor, vanadate, alone promoted PtdIns hydrolysis and acted non-additively with carbachol. Vanadate did not stimulate PtdInsP 2 breakdown. Carbachol also stimulated a rapid (maximal at 1-2 min) tyrosine phosphorylation of several islet proteins, although not of PLC␥1 itself. Two structurally unrelated inhibitors of PtdIns 3-kinase, wortmannin and LY294002, more effectively attenuated the hyrolysis of PtdIns compared with PtdInsP 2 . Adenovirally mediated overexpression of PLC␥1 significantly increased carbachol-stimulated PtdIns hydrolysis without affecting that of PtdInsP 2 . Conversely overexpression of PLC␤1 up-regulated the PtdInsP 2 , but not PtdIns, response. These results indicate that the hydrolysis of PtdIns and PtdInsP 2 are independently regulated in pancreatic islets and that PLC␥1 selectively mediates the breakdown of PtdIns. The activation mechanism of PLC␥ involves tyrosine phosphorylation (but not of PLC␥ directly) and PtdIns 3-kinase. Our findings point to a novel bifurcation of signaling pathways downstream of muscarinic receptors and suggest that hydrolysis of PtdIns and PtdInsP 2 might serve different physiological ends.Many receptor tyrosine kinases and GPCRs 1 couple to PLC that catalyzes the cleavage of PtdInsP 2 and resultant generation of the two second messengers Ins(1,4,5)P 3 , which mobilizes Ca 2ϩ from intracellular stores, and DAG, which activates protein kinase C (1-4). These pathways are well described in the regulation of insulin secretion following occupation of m3 and m1 muscarinic receptors on the surface of pancreatic ␤Ϫcells (5-8). However we have shown that in addition to the classical pathway of PtdInsP 2 hydrolysis, exposure of pancreatic islets to the muscarinic receptor agonist carbachol also promotes the hydrolysis of PtdIns (9, 10). The latter predominates quantitatively over, and is a precursor of, PtdInsP 2 . Although hydrolysis of either phosphoinositide species results in generation of DAG, only PtdInsP 2 gives rise to a Ca 2ϩ signal via Ins(1,4,5)P 3 production. Therefore the two hydrolytic events may have different functional consequences. The inositol phosphate, corresponding to Ins(1,4,5)P 3 , that is derived from PtdIns, is Ins(1)P 1 . This has no biological function but can be used to quantify PtdIns hydrolysis, at least under the conditions verified in our previous studies (9).The PLC family is comprised of four major groups (PLCs ␤, ␥, ␦, and ⑀). Members of the PLC␤ family are activated by Gproteins in response to o...