Erythrocytes have been reported to release ATP from intracellular stores into the surrounding environment in response to decreased oxygen tension and mechanical deformation. This erythrocyte-derived ATP can then act on purinergic receptors present on vascular endothelial cells, resulting in the synthesis and bidirectional release of nitric oxide (NO). NO released abluminally produces relaxation of vascular smooth muscle, thereby increasing vascular caliber, leading to a decrease in deformation-induced ATP release from erythrocytes. In contrast, NO released into the vascular lumen could interact directly with formed elements in the blood, including the erythrocyte. Here, we investigate the hypothesis that NO functions in a negative-feedback manner to inhibit ATP release from the erythrocyte. The NO donor N-(2-aminoethyl)- N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) decreased total pulmonary resistance in a dose-dependent manner when administered to isolated perfused rabbit lungs. ATP release from rabbit erythrocytes in response to decreased oxygen tension or mechanical deformation was inhibited by preincubation with spermine NONOate (100 nM, 20 min). Importantly, incubating rabbit erythrocytes with spermine (100 nM, 20 min), the polyamine remaining after the liberation of NO from spermine NONOate, did not affect decreased oxygen tension-induced ATP release. Mechanical deformation-induced ATP release was also inhibited when erythrocytes were preincubated with spermine NONOate. However, NO-depleted spermine NONOate had no effect on mechanical deformation-induced ATP release from rabbit erythrocytes. These data provide support for the hypothesis that NO inhibits ATP release from erythrocytes, thereby identifying an additional role of NO in the regulation of vascular resistance.