The hydrolysis of phosphoinositides by phospholipase C is a major pathway of signal transduction during the response of many cells to stimulation by hormones, peptide growth factors, neurotransmitters, and other regulatory ligands. (1) Three distinct phospholipase C isozymes (PLC-I, II, and III) were purified to homogeneity from bovine brain. Their apparent MW determined under a denaturing condition were 150,000, 145,000 and 85,000 for PLC-I, II, and III, respectively. PLC-I, and III mainly exist in dimeric form, whereas PLC-II is present predominantly as monomer. (2) The catalytic properties of the 3 isozymes were studied by using small unilamellar vesicles prepared from either phosphatidylinositol (PI) or phosphatidylinositol-4,5-bisphosphate (PI-bisphosphate) as substrate. Hydrolysis of both PI and PI-bisphosphate by the 3 enzymes was dependent on calcium. However, at low calcium PI as substrate, the order of specific activity was PLC-III greater than PLC-I. Contrarily, the order was PLC-I greater than PLC- III greater than PLC-II for PI-bisphosphate hydrolysis, meaning that PLC-I is the most specific for PI-bisphosphate. (3) Both polyclonal (rabbit) and murine monoclonal antibodies were prepared against the 3 isozymes. Antibodies derived against one form of the isozymes did not cross-react with another form, indicating that the 3 isozymes are immunologically independent. (4) The subcellular distribution of PLC-I and PLC-II in brain homogenates was measured using radioimmunoassay. More than 90% of PLC-II was found in the cytosolic fractions, whereas the PLC-I was equally distributed between cytosolic fractions. (5) The distribution of PLC-I and PLC-II was studied in 17 different rat organs and 20 different cultured cells. PLC-I is specific to brain whereas PLC-II is widely distributed in various tissues and cell. (6) Monoclonal antibodies were used to map the distribution of PLC-I and PLC-II in rat brain using immunohistochemical techniques. Both forms of PLC appear to be distributed widely but in specific neuronal systems. In some systems, both forms of the enzymes appear to be co-localized. However, in other systems, PLC-II appears to be expressed whereas PLC-I is not.