The most abundant and widely expressed mammalian phosphoinositide kinase activity is contributed by phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha). In this study we demonstrate that PI4KIIalpha is a novel GTP-independent target of the wasp venom tetradecapeptide mastoparan and that different mechanisms of activation occur in different subcellular membranes. Following cell membrane fractionation mastoparan specifically stimulated a high activity Golgi/endosomal pool of PI4KIIalpha independently of exogenous guanine nucleotides. Conversely, GTPgammaS stimulated a low activity pool of PI4KIIalpha in a separable dense membrane fraction and this response was further enhanced by mastoparan. Overexpression of PI4KIIalpha increased the basal phosphatidylinositol 4-kinase activity of each membrane pool, as well as the mastoparan-dependent activities, thereby demonstrating that mastoparan specifically activates this isozyme. Both mastoparan and M7, at concentrations known to invoke secretion, stimulated PI4KIIalpha with similar efficacies, resulting in an increase in the apparent V(max) and decrease in K(m) for exogenously added PI. Mastoparan also stimulated PI4KIIalpha immunoprecipitated from the raft fraction, indicating that PI4KIIalpha is a direct target of mastoparan. Finally we reveal a striking dependence of both basal and mastoparan-stimulated PI4KIIalpha activity on endogenous cholesterol concentration and therefore conclude that changes in membrane environment can regulate PI4KIIalpha activity.