NITRIC oxide level in the mouse brain was estimated by determination of nitrite and nitrate using an automated flow injection analyser for NOx. Different experimental conditions were examined to determine which produced reproducible results. After pretreatment of tissue specimens by the ZnSO4-NaOH method for deproteinization, reproducible and constant values were obtained. The values were more accurate immediately after sectioning without freezing than after 24 h with freezing. Two sacrifice methods, decapitation and microwave irradiation of the head, were investigated, but there was no significant difference between the two. No substances in the mouse brain exerted a positive or negative influence on the results. These results show that our method is indeed applicable to the brain tissue.