Hydrolysis of exogenous one-acyl,2~3H]-oleoy1 phosphatidylcholine by intracellular phospholipase A2 to yield free I3H1-oleate and lysophosphatidylcholine was measured in isolated liver microsomes from warm (20")and cold (5 "(2)-acclimated rainbow trout, Salmo gairdneri. Unlike mammalian phospholipase AZ, which is not typically microsomal, over 18% of the total phospholipase A2 activity from trout liver occurred in the microsomes. Specific activity in microsomal preparations (4.07 nmole * hr-' * mg-l) represented a 1.4-fold purification of clarified liver homogenates (2.93 nmole-hr-'mg-'). The accumulation of free fatty acid was a function of both the duration and the protein concentration of the assay. Activity was enhanced fourfold in the presence of 0.1% Triton X-100. Free C a + + stimulated activity up to 100 pM, but was not an absolute requirement of the enzyme. Maximal activity occurred at pH 8.0-8.5 at 20ยฐC assay temperature and pH 9.0 at 5ยฐC. Acclimation history did not influence the pH optima of the enzyme. Thermal acclimation did, however, result in perfect compensation of enzymatic rate; phospholipase A2 activity from cold-acclimated trout was not significantly different from that of warm-acclimated trout when measurements were made at their respective acclimation temperatures. This perfect compensation for temperature as well as the unique intracellular localization of trout liver phospholipase A2 implicate the enzyme in a deacylation-reacylation cycle that could be partially responsible for the thermally modulated restructuring previously observed in microsomal membranes of this species.