We previously reported that cytosolic fractions of bovine brain contain two immunologically distinct forms of phospholipase C (PLC), PLC-I and PLC-II. We now report the purification of another form of inositolphospholipid-specific phospholipase C from bovine brain cytosol, designated PLC-III, and the comparison of the catalytic properties of the three isozymes. Approximately 450 micrograms of pure PLC-III was obtained from 36 bovine brains, and it had a final specific activity of 30-40 mumol of phosphatidylinositol hydrolyzed per min per mg of enzyme in the presence of 0.1% deoxycholate. PLC-III exhibited an apparent Mr of 85,000 in NaDodSO4/PAGE, which is considerably smaller than the Mr of 150,000 for PLC-I and 145,000 for PLC-II. Neither of the two mixtures of monoclonal antibodies nor the rabbit polyclonal antibodies directed against either PLC-I or PLC-II cross-reacted with PLC-III. The catalytic properties of the three isozymes were studied by using small unilamellar vesicles prepared from either phosphatidylinositol (PtdIns) or phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates. Hydrolysis of both PtdIns and PtdInsP2 by the three enzymes was dependent on Ca2+. However, at low Ca2+ concentration, PtdInsP2 was the preferred substrate for all three enzymes. When PtdIns was the substrate, the three enzymes exhibited similar specific activities at their optimum pH, which was 4.8 for PLC-I, 5.0 for PLC-II, and 5.5 for PLC-III. But at neutral pH, the order of specific activity was PLC-III greater than PLC-II greater than PLC-I. In contrast, the order of specific activity was PLC-I greater than PLC-III greater than PLC-II for PtdInsP2 hydrolysis, which means that PLC-I is the most specific for PtdInsP2. The three enzymes were affected differently by bovine serum albumin: inhibition of PLC-I and activation of PLC-III were observed, whereas PLC-II was unaffected. This observation suggests that any putative protein effectors for PLC should be critically scrutinized.