In the previous paper1), we have assumed that nitrite ion was not produced from nitro-glycol in vivo, and that methemoglobin found in the blood of rabbits injected with nitro-glycol was formed by the action of nitroglycol, but not by nitrite. In this paper, thisassumption was ascertained. When rabbit received nitroglycol subcutaneously, a largeamount of free nitroglycol was found in the blood immediately after the injection. Theamount of methemoglobin found in the blood was in parallel with the amount of freenitroglycol in the blood. As a metabolic product of nitroglycol, nitrate ion was detectedin the blood, but nitrite was not. When nitroglycol was injected daily, the rate of nitrateproduction increased day after day. This phenomenon was interpreted as being due tothe increment of nitroglycol hydrolyzing enzyme.