Current studies involve an investigation of the role of the pleckstrin homology (PH) domain in membrane targeting and activation of phospholipase Cbeta(1) (PLCbeta(1)). Here we report studies on the membrane localization of the isolated PH domain from the amino terminus of PLCbeta(1) (PLCbeta(1)-PH) using fluorescence microscopy of a green fluorescent protein fusion protein. Whereas PLCbeta(1)-PH does not localize to the plasma membrane in serum-starved cells, it undergoes a rapid but transient migration to the plasma membrane upon stimulation of cells with serum or lysophosphatidic acid (LPA). Regulation of the plasma membrane localization of PLCbeta(1)-PH by phosphoinositides was also investigated. PLCbeta(1)-PH was found to bind phosphatidylinositol 3-phosphate most strongly, whereas other phosphoinositides were bound with lower affinity. The plasma membrane localization of PLCbeta(1)-PH induced by serum and LPA was blocked by wortmannin pretreatment and by LY294002. In parallel, activation of PLCbeta by LPA was inhibited by wortmannin, by LY294002, or by the overexpression of PLCbeta(1)-PH. Microinjection of betagamma subunits of G proteins in serum-starved cells induced the translocation of PLCbeta(1)-PH to the plasma membrane. These results demonstrate that a cooperative mechanism involving phosphatidylinositol 3-phosphate and the Gbetagamma subunit regulates the plasma membrane localization and activation of PLCbeta(1)-PH.