Expression of endothelial nitric oxide synthase (eNOS) in transfected U-937 cells upregulates phorbol 12-myristate 13-acetate (PMA)-induced tumor necrosis factor- (TNF-) production through a superoxide (O)-dependent mechanism. Because mitogen-activated protein kinases (MAPK) have been shown to participate in both reactive oxygen species signaling and TNF- regulation, their possible role in eNOS-derived O signal transduction was examined. A redox-cycling agent, phenazine methosulfate, was found to both upregulate TNF- (5.8 ± 1.0 fold; P = 0.01) and increase the phosphorylation state of p42/44 MAPK (3.1 ± 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. Although S-nitroso-N-acetylpenicillamine, a nitric oxide (NO) donor, also increased TNF- production, NO exposure led to phosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF- production by eNOS transfection was associated with increases in activated p42/44 MAPK (P = 0.001), whereas levels of phosphorylated p38 MAPK were unaffected. Furthermore, cotransfection with Cu/Zn superoxide dismutase, which blocks TNF- upregulation by eNOS, also abolished the effects on p42/44 MAPK. Expression of Gln³⁶¹eNOS, a mutant that produces O but not NO, still resulted in p42/44 MAPK phosphorylation. In contrast, two NADPH binding site deletion mutants of eNOS that lack oxidase activity had no effect on p42/44 MAPK. Finally, PD-98059, a p42/44 MAPK pathway inhibitor, blocked TNF- upregulation by eNOS (P = 0.02). Thus O produced by eNOS increases TNF- production via a mechanism that involves p42/44 MAPK activation.