2-Nitropropane (2-NP), a rat hepatocarcinogen, is denitrified to nitrite and acetone by rat liver microsomes; the denitrification rate is increased using microsomes from phenobarbital (PB)-pretreated rats. To obtain evidence that denitrification of 2-NP also occurs in vivo, we attempted to determine nitrite and nitrate levels in blood sera and urines of 2-NP-treated (1.5 mmol/kg, ip, once) rats with and without PB pretreatment (80 mg/kg, ip, once daily, 3 days), using enzymatic reduction followed by the standard Griess reaction. However, due to various interfering factors, including pigment from methemoglobinemia, we found the assay had to be modified as follows: (a) reduction of nitrate to nitrite was accomplished using NADPH and nitrate reductase, (b) excess NADPH, proteins, and interfering pigments were precipitated using zinc acetate and Na 2 CO 3 , and (c) the Griess reagents were prepared in 3 N HCl rather than 5% H 3 PO 4 . With these modifications it became possible to show that 2-NP is indeed metabolized to nitrite in vivo and that the metabolism is increased by PB pretreatment. Two hours after 2-NP administration, rat blood serum nitrate plus nitrite levels were approximately 1600 μM (PB-pretreated) and 940 μM (vehicle-pretreated controls). The PB-pretreated and control rats, respectively, excreted 250 and 120 μmol nitrate/nitrite in the 24-h urine post 2-NP treatment. The modifications described make the method more specific, reproducible, and more widely applicable.