1. Glycerol trinitrate (GTN) has been used in therapy for more than 100 years. Biological effects of GTN are due to the release of the biomediator nitric oxide (NO). However, the mechanism by which GTN provides NO, in particular in liver, is still unknown. In this study, we provide experimental evidence showing that cytoplasm, endoplasmic reticulum, and mitochondria are required for the release of NO from GTN in the liver. 2. NO and nitrite (NO(2)(-)) were determined using low-temperature electron paramagnetic resonance and the Griess reaction, respectively. 3. The first step of GTN biotransformation is the release of NO(2)(-). This step is performed in cytoplasm and catalyzed by glutathione-S-transferase. The second step is the rate-limiting step where NO(2)(-) is slowly reduced to NO. This is mainly catalyzed by cytochrome P-450. The second phase can be significantly enhanced by decreasing the pH value, a situation which occurs during ischemia. At high NADPH concentrations exceeding physiological values, cytochrome P-450 catalyzes GTN biotransformation without the involvement of cytoplasmic glutathione-S-transferase. 4. In conclusion, our data show that NO(2)(-) derived from the first step of biotransformation of GTN in the liver is the precursor of NO but not a product of NO degradation; consequently, NO(2)(-) levels are not likely to be a marker of NO release from GTN as earlier suggested.