In contrast, the combined action of the two enzymes resulted in a highly significant dose-dependent and time-dependent loss of surfactant function, concomitant with PC and LPC hydrolysis and the generation of free PA. PLA2 alone was insufficient to generate a loss of surfactant function, possibly because the LPC product functionally replaces the lost PC. However, with the hydrolysis catalyzed by the eosinophil LPLase, the LPC concentration diminished significantly, likely leading to the loss of surfactant activity. Thus, the critical enzyme in this system is the 25-kd eosinophil LPLase. Equivalent results were obtained using whole-cell lysates of an eosinophil cell line (AML14.3D10) in the absence of exogenously added PLA2, suggesting that the eosinophil expresses all of the necessary lipolytic activities. The inhibition of the LPLase activity in AML14.3D10 eosinophil extracts by a non-selective, sulfhydryl-reactive, ser/thr protease inhibitor, N-ethylmaleimide, reduced its ability to decrease surfactant activity.