To elucidate the dynamics of nitric oxide (NO) metabolism in the circulation and its relationship with glutathione metabolism, formation of nitrosylhemoglobin (NO-Hb), S-nitrosothiols (RSNO), and nitrite+nitrate (NOx) was determined in blood samples from normal rats and animals that were treated with a loading dose of GSH or L-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of GSH synthesis. When incubated in vitro with 0.2 mM NOC7, an NO donor, NO-Hb levels increased rapidly, peaked at 10 min, and decreased thereafter with a half-life of 35 min in blood samples from control, BSO-treated, or GSH-loaded animals. Levels of low-molecular-weight RSNO in plasma samples from the three animal groups also increased transiently, peaked at 10 min, and decreased thereafter. However, the amount of RSNO formed in GSH-loaded rat plasma was significantly greater than in control and BSO-treated animals. Plasma levels of NOx rapidly and similarly increased in all animal groups. Intravenously injected NOC7 also generated NO-Hb in circulating erythrocytes. In control animals, blood levels of NO-Hb increased maximally at 30 min and decreased thereafter with a half-life of 100 min. NO-Hb formed in the GSH-loaded group was significantly lower than in the control group. In contrast, the rate of NO-Hb formation was significantly higher with the BSO-treated group than with the control group. Although NOC7 did not affect the plasma levels of low-molecular-weight RSNO in plasma of both control and BSO-treated groups, it significantly increased RSNO in the GSH-loaded group. Thirty minutes after administration of NOC7, about 20% of the dose was recovered as plasma NOx in all animal groups. These results suggested that GSH status in animals might affect the metabolism of NO.