The effect of phosphoinositides on the activity of protein kinase C (PKC) isotypes was investigated. PKC alpha, beta I, beta II, gamma, delta, epsilon, eta, and zeta were expressed in baculovirus-infected insect cells and purified by column chromatography. The calcium-activated PKC isotypes alpha, beta I, beta II, and gamma were not significantly activated by any of the phosphoinositides investigated (phosphatidylinositol-4-phosphate (PtdIns-4-P), PtdIns-3-P, PtdIns-4,5-P2, PtdIns-3,4-P2, and PtdIns-3,4,5-P3) when added in the presence of concentrations of phosphatidylserine that give maximal stimulation. The calcium-insensitive PKC isotypes delta, epsilon, and theta also showed little response to PtdIns-3-P, PtdIns-4-P, or PtdIns-4,5-P2 when these lipids were added in the presence of phosphatidylserine. In contrast, PtdIns-3,4-P2 and PtdIns-3,4,5-P3 caused a 5-15-fold stimulation of these enzymes compared with phosphatidylserine alone. 50% maximal stimulation of PKC epsilon by PtdIns-3,4,5-P3 occurred when this lipid was present at about 1% of the carrier PtdIns-4,5-P2 (about 100 nM). These lipids had little effect on baculovirus-expressed PKC zeta, which was constitutively active. A short chain version of PtdIns-3,4,5-P3, dioctanoyl-PtdIns-3,4,5-P3, activated PKC delta, epsilon, and eta in the absence of other lipids, whereas a short chain version of PtdIns-4,5-P2, dihexanoyl-PtdIns-4,5-P2, did not. Since PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are nominally absent in unstimulated cells and appear within seconds to minutes of stimulation by various cell activators, these lipids could act as second messengers to activate PKC delta, epsilon, or eta in vivo.