Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.