Surfactin inhibits platelet and spleen cytosolic 100 kDa phospholipase A 2 (PLA 2). In contrast, this same compound enhances rat platelet group II PLA 2 activity by ∼2-fold and slightly increases group I PLA 2 activity from porcine pancreas and Naja naja venom in vitro. Surfactin does not affect a Ca 2+-independent PLA 2 partially purified from bovine brain. Thus, this compound inhibits selectively the cytosolic form of PLA 2. Based on in vitro studies utilizing preincubation of surfactin with the enzyme, dialysis, and increased concentrations of substrates, the inhibitory effect of surfactin appears to be due to a direct interaction with the enzyme. Linear regression analysis of the linear portion of a concentration–response curve reveals an ic 50 of 8.5 μM. To further determine the inhibitory pattern, a Dixon plot was constructed to show that the inhibition by surfactin is competitive, but not uncompetitive, with an inhibition constant of K i = 4.7 μM in 50 mM Tris–HCl buffer, pH 8.0, at 37°. Surfactin blocked non-stimulated and calcium ionophore A23187-stimulated release of arachidonic acid from monkey kidney CV-1 cells, which contain a cytosolic 100 kDa PLA 2 as the major activity, as shown in an anionic exchange DEAE-5PW high performance liquid chromatography profile and western blotting analysis. Surfactin ameliorated inflammation induced by several chemicals. That is, it exhibited in vivo anti-inflammatory activity in several tested inflammatory reactions including 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, carrageenan-induced rat paw edema, and acetic acid-induced mouse writhing. These results demonstrate that surfactin is a selective inhibitor for cytosolic PLA 2 and a putative anti-inflammatory agent through the inhibitory effect produced by direct interaction with cytosolic PLA 2, and that inhibition of cytosolic PLA 2 activity may suppress inflammatory responses.