Analysis of purified soybean and rabbit reticulocyte 15-lipoxygenase (15-LOX) and PA317 cells transfected with human 15-LOX revealed a rapid rate of linoleate-dependent nitric oxide (·NO) uptake that coincided with reversible inhibition of product ((13S)-hydroperoxyoctadecadienoic acid, or (13S)-HPODE) formation. No reaction of ·NO (up to 2 μM) with either native (E(red)) or ferric LOXs (0.2 μM) metal centers to form nitrosyl complexes occurred at these ·NO concentrations. During HPODE-dependent activation of 15-LOX, there was consumption of 2 mol of ·NO/mol of 15-LOX. Stopped flow fluorescence spectroscopy showed that ·NO (2.2 μM) did not alter the rate or extent of (13S)-HPODE-induced tryptophan fluorescence quenching associated with 15-LOX activation. Additionally, ·NO does not inhibit the anaerobic peroxidase activity of 15-LOX, inferring that the inhibitory actions of ·NO are due to reaction with the enzyme-bound lipid peroxyl radical, rather than impairment of (13S)-HPODE-dependent enzyme activation. From this, a mechanism of 15-LOX inhibition by ·NO is proposed whereby reaction of ·NO with E(red)LOO· generates E(red) and LOONO, which hydrolyzes to (13S)-HPODE and nitrite (NO2/-). Reactivation of E(red), considerably slower than dioxygenase activity, is then required to complete the catalytic cycle and leads to a net inhibition of rates of (13S)-HPODE formation. This reaction of ·NO with 15-LOX inhibited ·NO-dependent activation of soluble guanylate cyclase and consequent cGMP production. Since accelerated ·NO production, enhanced 15-LOX gene expression, and 15-LOX product formation occurs in diverse inflammatory conditions, these observations indicate that reactions of ·NO with lipoxygenase peroxyl radical intermediates will result in modulation of both ·NO bioavailability and rates of production of lipid signaling mediators.