Evidence accumulated to date suggests that extracellular group II phospholipase A2 (PLA2-II) is involved in the pathogenesis of inflammatory disease. During screening for PLA2 inhibitors, we found a novel PLA2 inhibitor named thielocin B3 in the culture broth of an ascomycetes. Thielocin B3 strongly inhibited human PLA2-II (IC50 = 0.076 microM) in a reversible and noncompetitive manner (Ki = 0.098 microM), whereas it inhibited human group I PLA2 only weakly (IC50 = 18 microM). It also quenched the tryptophan fluorescence of Naja mocambique venom PLA2; almost 100% quenching being attained at a thielocin B3/enzyme molar ratio of 1.0. Its inhibitory activity toward human PLA2-II and Naja mocambique PLA2 was markedly decreased by methylation of its two carboxyl groups, while the quenching observed for Naja mocambique PLA2 was not altered. These results suggest that the two carboxyl groups do not participate in the binding of thielocin B3 to the enzyme, but play a crucial role in the PLA2 inhibition. Furthermore, in the rat carrageenan-induced pleurisy model, thielocin B3 significantly reduced both exudate volume and PLA2 activity in the exudate when coinjected with carrageenan.