Nitric oxide generation is involved in a range of diseases involving polymorphonuclear leukocytes. The aim of this study was to determine whether human polymorphonuclear leukocytes are able to generate nitric oxide and to investigate the time course of its generation after stimulation with 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, 60 ng/ml phorbol myristate acetate, 10(-7) M concanavalin A, and 10(-7) M platelet activating factor. Stimulation of human polymorphonuclear leukocytes with N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate caused sustained nitric oxide generation, reaching maximal values of 1,105 +/- 361 nM (n = 32) and 628 +/- 119 nM (n = 30), respectively. Platelet activating factor did not affect nitric oxide production (maximal value 29 +/- 7 nM, n = 8), whereas concanavalin A caused only a slight increase (102 +/- 24 nM, n = 8) when compared with resting cells control (26 +/- 6 nM, n = 8). Human polymorphonuclear leukocytes were able to respond to both consecutive and alternate N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate stimulation with nitric oxide generation. Nitric oxide generation was inhibited by specific inhibitors (N omega-nitro-L-arginine and N omega-monomethyl-L-arginine) and restored with L-arginine. We provide, to our knowledge, the first direct evidence that human neutrophils generate nitric oxide.