Methyl arachidonyl fluorophosphonate (MAFP) has been recently reported to be a selective, active-site directed, irreversible inhibitor of the Group IV 85 kDa cytosolic phospholipase A2 (cPLA2). We have now shown that this compound also potently inhibits the Ca(2+)-independent cytosolic phospholipase A2 (iPLA2). MAFP inhibited iPLA2 in a concentration-dependent manner with half-maximal inhibition observed at 0.5 microM after a 5 min preincubation at 40 degrees C. This inhibition was not reversed upon extensive dilution of the enzyme into the assay mixture. Preincubation of iPLA2 with MAFP resulted in a linear, time-dependent inactivation of enzyme activity, and the enzyme was protected from inactivation by the reversible inhibitor PACOCF3. The ability of MAFP to inhibit the iPLA2 suggests that this enzyme proceeds through an acyl-enzyme intermediate as has been proposed for the cPLA2. Further testing indicated that MAFP did not inhibit the arachidonoyl-CoA synthetase, CoA-dependent acyltransferase, or CoA-independent transacylase activities from P388D1 cells. Thus, MAFP is not a general inhibitor for enzymes which act on arachidonoyl substrates. Instead, the inhibitor appears to show some selectivity for PLA2, although it does not discriminate between cPLA2 and iPLA2. Particular caution must be exercised to distinguish these activities if this inhibitor is used in intact cells.