Endothelium-derived nitric oxide (NO) inhibits in vitro platelet aggregation via a cGMP-dependent mechanism. The effect of inhaled NO on platelet-mediated pulmonary thrombosis following intravenous thrombotic challenge with collagen was examined in rats and compared with the effect of G4120, a cyclic Arg-Gly-Asp-containing synthetic pentapeptide that binds to the platelet glycoprotein IIb/IIIa receptor. Intraplatelet cGMP dose-dependently increased from 39 +/- 6 fmol/10(8) platelets in control to 46 +/- 6, 68 +/- 13, and 81 +/- 13 fmol/10(8) platelets after inhalation with 20, 40, and 80 ppm NO, respectively (P < .05 for 40 and 80 ppm). Ex vivo platelet aggregation of platelet-rich plasma induced by 1 microgram/mL collagen was reduced from 75 +/- 4% in control rats to 22 +/- 10% and 20 +/- 7% in rats ventilated with 40 and 80 ppm NO, respectively, and to 30 +/- 9% in G4120-treated rats (each P < .05 versus control). Circulating platelet counts 3 minutes after collagen injection were significantly higher in the inhaled NO and G4120 groups compared with control rats (250,000 +/- 18,000 and 223,000 +/- 10,000/microL versus 160,000 +/- 18,000/microL, each P < .05). The rise in pulmonary arterial pressure after collagen injection was significantly reduced in NO- and G4120-treated rats (26 +/- 1 and 27 +/- 1 versus 32 +/- 1 mm Hg in control rats, each P < .05). The number of pulmonary resistance vessels containing platelet thrombi was significantly smaller after inhaled NO and G4120 treatment compared with control (56 +/- 3% and 50 +/- 3% versus 68 +/- 3%, respectively; P < .05). Thus, NO inhalation reduces in vivo activation of circulating platelets and platelet-rich thrombosis in thromboembolic pulmonary hypertension. Inhalation of NO may be useful in cardiovascular diseases associated with platelet activation.