The pharmacokinetics and metabolism of pyrazine-2-diazohydroxide have been studied in the beagle dog and mouse. When pyrazine-2-diazohydroxide was administered to beagle dogs at a dose of 18.6 mg/kg (428 mg/m2) by i.v. bolus, the plasma half-life (t1/2) was 7.3 min, the apparent volume of distribution (Vd) 577 ml/kg, and the total body clearance (Cl) 55 ml/min per kg. In mice given pyrazine-2-diazohydroxide by i.v. bolus at 100 mg/kg (428 mg/m2), the t1/2 was 5.8 min, the Vd 250 ml/kg, and the Cl 30 ml/min per kg. When [2-14C]pyrazine-2-diazohydroxide was infused i.v. to mice at 100 mg/kg over 8 h, the Cl for parent drug was 122 ml/min per kg. The major product formed from pyrazine-2-diazohydroxide was 2-hydroxypyrazine, which accounted for 80% of the total radioactivity in the plasma after a 6-h drug infusion. There were three other metabolites in plasma, two more polar than pyrazine-2-diazohydroxide, which accounted for 7% of the radioactivity, and one less polar, which accounted for 5% of the radioactivity. Following an i.v. bolus dose of [2-14C]pyrazine-2-diazohydroxide, 79% of the radioactivity was excreted in the urine in 24 h, 3% in the feces, and 0.4% in the expired air; 18% remained in the carcass. The liver and kidney showed the highest tissue levels of radioactivity. 2-Hydroxypyrazine accounted for 45% of the urinary radioactivity, pyrazine-2-diazohydroxide for 14%, and a glucuronide or sulfate conjugate of 2-hydroxypyrazine for 17%. Twenty-four percent of the radioactivity eluted near the void volume on high-performance liquid chromatography and was not identified.