We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-β1 and -β2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10-3 M. PLC-β1 and PLC-β2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10-5 M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-β1 and PLC-β2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein α- and βγ-subunit activators in the phospholipid vesicles does not increase the binding of PLC-β1 or PLC-β2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10-6 M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10-3 M). PLC-β1 and -β2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.