Treatment of human polymorphonuclear leukocytes (PMNLs) with micromolar concentrations of the anti-inflammatory drug manoalide inhibited production of leukotriene B4 (LTB4) and LTC4/LTD4 in response to the calcium ionophore A23187. In an attempt to further define the mechanism(s) of action of this agent, we have examined its interaction with several lipoxygenase enzymes. In RBL-1 cells, manoalide inhibited 5-lipoxygenase (5-LO) activity with an approximate IC50 of 0.3 microM. This was equipotent in our system with the known lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Manoalide was virtually inactive, however, against 12-lipoxygenase activity in both human platelets and mouse epidermis, with little inhibition seen at concentrations up to 100 microM. Manoalide showed some activity against soybean lipoxygenase, although it was 30- to 50-fold less potent than as an inhibitor of the 5-lipoxygenase enzyme. These data indicate that manoalide is a selective 5-LO inhibitor and suggest the possibility that its anti-inflammatory actions may be due, at least in part, to inhibition of leukotriene synthesis.