Herein, we demonstrate that nitric oxide is a potent (>20% release) and highly selective inducer of [3H]arachidonic acid mobilization in the macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one resulted in the inhibition of the large majority (86%) of nitric oxide-induced [3H]arachidonic acid release into the medium (IC50 <0.5 μM) and the concomitant inhibition of in vitro measurable calcium-independent phospholipase A2 activity (92% inhibition) without demonstrable effects on calcium-dependent phospholipase A2 activity. Since nitric oxide is a potent stimulator of glycolysis (and therefore glycolytically derived ATP) and since cytosolic calcium-independent phospholipase A2 exists as a catalytic complex comprised of ATP-modulated phosphofructokinase-like regulatory polypeptides and a catalytic subunit, we examined the role of glucose in facilitating nitric oxide-mediated arachidonic acid release. Nitric oxide-induced release of [3H]arachidonic acid possessed an obligatory requirement for glucose, was highly correlated with the concentration of glucose in the medium, and was dependent on the metabolism of glucose. Thus, [3H] arachidonic acid release is coupled to cellular glucose metabolism through alterations in the activity of calcium-independent phospholipase A2. Collectively, these results identify a unifying metabolic paradigm in which the generation of lipid second messengers is coordinately linked to the signalstimulated acceleration of glycolytic flux, thereby facilitating integrated metabolic responses to cellular stimuli.