1 The aim of this study was to establish the role of nitric oxide (NO) and cyclic GMP in chemotaxis and superoxide anion generation (SAG) by human neutrophils, by use of selective inhibitors of NO and cyclic GMP pathways. In addition, inhibition of neutrophil chemotaxis by NO releasing compounds and increases in neutrophil nitrate/nitrite and cyclic GMP levels were examined. The ultimate aim of this work was to resolve the paradox that NO both activates and inhibits human neutrophils. 2 A role for NO as a mediator of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis was supported by the ®nding that the NO synthase (NOS) inhibitor L-NMMA (500 mM) inhibited chemotaxis; EC 50 for fMLP 28.76+5.62 and 41.13+4.77 pmol/10 6 cells with and without L-NMMA, respectively. Similarly the NO scavenger carboxy-PTIO (100 mM) inhibited chemotaxis; EC 50 for fMLP 19.71+4.23 and 31.68+8.50 pmol/10 6 cells with and without carboxy-PTIO, respectively. 3 A role for cyclic GMP as a mediator of chemotaxis was supported by the ®nding that the guanylyl cyclase inhibitor LY 83583 (100 mM) completely inhibited chemotaxis and suppressed the maximal response; EC 50 for fMLP 32.53+11.18 and 85.21+15.14 pmol/10 6 cells with and without LY 83583, respectively. The same pattern of inhibition was observed with the G-kinase inhibitor KT 5823 (10 mM); EC 50 for fMLP 32.16+11.35 and 4135 pmol/10 6 cells with and without KT 5823, respectively. 4 The phosphatase inhibitor, 2,3-diphosphoglyceric acid (DPG) (100 mM) which inhibits phospholipase D, attenuated fMLP-induced chemotaxis; EC 50 for fMLP 19.15+4.36 and 61.52+16.2 pmol/10 6 cells with and without DPG, respectively. 5 Although the NOS inhibitors L-NMMA and L-canavanine (500 mM) failed to inhibit fMLP-induced SAG, carboxy-PTIO caused signi®cant inhibition (EC 50 for fMLP 36.15+7.43 and 86.31+14.06 nM and reduced the maximal response from 22.14+1.5 to 9.8+1.6 nmol O 2 7 /10 6 cells/10 min with and without carboxy-PTIO, respectively). This suggests NO is a mediator of fMLP-induced SAG. 6 A role for cyclic GMP as a mediator of SAG was supported by the e ects of G-kinase inhibitors KT 5823 (10 mM) and Rp-8-pCPT-cGMPS (100 mM) which inhibited SAG giving EC 50 for fMLP of 36.26+8.77 and 200.01+43.26 nM with and without KT 5823, and 28.35+10.8 and 49.25+16.79 nM with and without Rp-8-pCTP-cGMPS. 7 The phosphatase inhibitor DPG (500 mM) inhibited SAG; EC 50 for fMLP 33.93+4.23 and 61.12+14.43 nM with and without DPG, respectively. 8 The NO releasing compounds inhibited fMLP-induced chemotaxis with a rank order of potency of GEA 3162 (IC 50 =14.72+1.6 mM)4GEA 5024 (IC 50 =18.44+0.43 mM)4SIN-1 (IC 50 41000 mM). This order of potency correlated with their ability to increase cyclic GMP levels rather than the release of NO, where SIN-1 was most e ective (SIN-1 (EC 50 =37.62+0.9 mM)4GEA 3162 (EC 50 =39.7+0.53 mM)4 GEA 5024 (EC 50 =89.86+1.62 mM)). 9 In conclusion, chemotaxis and SAG induced by fMLP can be attenuated by inhibitors of phospholipase D, NO and cyclic GMP, suggesting a role for the...