Dipalmitoyl phosphatidylcholine (deltaPC) synthesis by lung epithelium occurs in part by a deacylation/reacylation pathway utilizing phospholipase A2 (PLA2) and an acyl transferase. The role of acidic Ca2+-independent PLA2 (aiPLA2) in this pathway was investigated using a transition-state analog enzyme inhibitor [1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33)]. Granular pneumocytes were isolated from rat lung with elastase and were maintained in primary culture for 24 h on microporous membranes in the presence of radiolabeled choline or free fatty acids (palmitate plus oleate). Disaturated phosphatidylcholine (DSPC) was determined by osmication chromatography. Incorporation (nmol/mg protein) into DSPC at 24 h incubation was 11.9 +/- 0.2 for [3H]choline and 12.1 +/- 0.04 for [3H]palmitate. In the presence of 3 mol% MJ33, incorporation of [3H] choline and [3H]palmitate was decreased by 37 and 69%, respectively, and DSPC pool size (microg/mg cell protein) decreased by 9% (P < 0.05). A similar decrease in radiolabel incorporation was observed with 2 h of incubation. The presence of p-bromophenacyl bromide (20 microm) had a significantly smaller effect that was additive with that of MJ33. After 24 h of labeling and 4 h of chase with unlabeled substrate, there was a significant decrease of radiolabel in DSPC that was inhibited by MJ33. Under all experimental conditions, MJ33 resulted in either no change or a modest increase of radiolabel in the cellular unsaturated PC fraction. These results indicate that aiPLA2 has a major role in DSPC synthesis by granular pneumocytes.