Activation of p85/p110-type phosphatidylinositol (PI) kinase has been implicated in various cellular activities. This PI kinase phosphorylates the D-4 position with a similar or higher efficiency than the D-3 position when trichloroacetic acid-treated cell membrane is used as a substrate, although it phosphorylates almost exclusively the D-3 position of the inositol ring in phosphoinositides when purified PI is used as a substrate. Furthermore, the lipid kinase activities of p110 for both the D-3 and D-4 positions were completely abolished by introducing kinase-dead point mutations in their lipid kinase domains (ΔKinα and δKinβ, respectively). In addition, both PI 3- and PI 4-kinase activities of pl10α and pl101β immunoprecipitates were similarly inhibited by either wortmannin or LY294002, specific inhibitors of pl10. Insulin induced phosphorylation of not only the D-3 position, but also the D-4 position. Indeed, overexpression of pl10 in Sf9 or 3T3-L1 cells induced marked phosphorylation of the D-4 position to a level comparable to or much greater than that of D-3, whereas inhibition of endogenous p85/pl10-type PI kinase via overexpression of dominant-negative p85α (Δp85α) in 3T3-L1 adipocytes abolished insulin-induced synthesis of both. Thus, p85/pl10-type PI kinase phosphorylates the D-4 position of phosphoinositides more efficiently than the D-3 position in vivo, and each of the D-3- or D-4-phosphorylated phosphoinositides may transmit signals downstream.