The enzyme phospholipase A2 (PLA2) catalyzes phospholipid hydrolysis and is thought to play important roles in surfactant synthesis and in the generation of lipid mediators, including eicosanoids and platelet-activating factor. This study sought to characterize PLA2 in rat type II pneumocytes by biochemical and immunologic means. Type II cells were found to contain an alkaline-active, Ca(2+)-dependent PLA2 activity predominantly localized to cytosol fraction. This activity preferred phospholipids containing arachidonate in the sn-2 position and phosphatidylethanolamine (PE) over phosphatidylcholine (PC); it was active at physiologically relevant Ca2+ concentrations and was resistant to dithiothreitol. These biochemical features were suggestive of a high-molecular-weight form of PLA2, and immunoblot analysis of type II cell extracts indeed detected a 97-kDa protein corresponding to a high-molecular-weight PLA2, but no low-molecular-weight pancreatic-type enzyme. Human A549 cells, which share certain features with type II cells, contained a similar PLA2 activity and immunoreactive protein. By contrast, whole rat lung cytosol contained both a 97-kDa PLA2 and a 14-kDa pancreatic-type PLA2, but its activity was typical of the latter in that it lacked sn-2 specificity for arachidonate and was inhibited by dithiothreitol. These results indicate that rat type II as well as human A549 epithelial cells contain a high-molecular-weight PLA2, for which mounting evidence suggests an important role in physiological and pathophysiological situations.