The role of phospholipase A2 (PLA2) in the simultaneous generation of lyso-platelet-activating factor and arachidonic acid was investigated by examining the calcium dependency and substrate specificity of PLA2 activities in rabbit lung microsomes. Alkylarachidonoylglycerophosphocholine (alkylarachidonoyl-GPC) was preferentially hydrolyzed as compared to acylarachidonoyl-GPC, and both arachidonate-containing substrates were cleaved to a greater extent as compared to alkyl- and acyl-substrates with oleate at the sn-2 position. Hydrolysis of alkylacyl-GPC substrates was not dependent on calcium in the presence of EGTA (1 mM); however, addition of calcium (2 mM) increased hydrolysis of acylarachidonoyl-GPC 2-fold and hydrolysis of acyloleoyl-GPC 10-fold. Substitution of an alkenyl group in the sn-1 position further enhanced calcium-independent PLA2 hydrolysis, and another substitution of arachidonic acid at the sn-2 position of the plasmalogen substrates substantially increased hydrolysis as compared to hydrolysis of substrates containing oleic acid. Hydrolysis of the choline plasmalogen was 3-fold greater than hydrolysis of the ethanolamine plasmalogen containing arachidonate. Preferential calcium-independent hydrolysis of alkylacyl-GPC substrates was observed in several tissues, including adult and fetal rabbit lung and adult rabbit kidney and human amnion. PLA2 substrate specificity may account for the preferential hydrolysis of arachidonoyl-containing alkyl-GPC in several cell types and explain the simultaneous generation of the precursors of two potent autacoids, platelet-activating factor and eicosanoids.