Plasma membranes were isolated from lapine synovial cells grown in monolayer culture using discontinuous sucrose gradient centrifugation techniques. 5'nucleotidase was detected in great abundance while glucose-6 phosphate dehydrogenase and cytochrome oxidase were present at low to undetectable levels. Plasma membranes incubated at 37 degrees C for 60 min with [3H]-arachidonyl-phosphatidylinositol/phosphatidylserine synthesized [3H]-diacylglycerides. Little if any [3H]-diacylglyceride synthesis was measured when [3H]-arachidonyl-phosphatidylcholine or [3H]-arachidonyl-phosphatidylethanolamine were used as substrates. These results are consistent with a plasma membrane-associated phosphatidylinositol-specific phospholipase C from lapine synovial cells in culture.