We reported previously that mice exposed to atmospheric NO2 contained a nitrosating agent (NSA) that reacted with morpholine in aqueous methanol homogenates of the mice to give N-nitrosomorpholine. We have now found that N-nitrosomorpholine was also produced by reacting morpholine with ether extracts of aqueous homogenates prepared from NO2-exposed mice. After exposure to NO2 for 4 hr, mice contained NSA (5.0 nmol/g tissue, corrected to 50 ppm NO2 and assuming that 1 mol NSA yields 1 mol N-nitrosomorpholine). This is 3.6 times the concentration observed by our previous method. Some NSA (0.6 nmol/g tissue) was also detected in untreated mice. The NSA in ether extracts was nonvolatile and stable on storage at -15 degrees or for short periods in the presence of water at pH 1 to 10, but it was decomposed by a pH 1 solution of nitrite scavengers. It reacted to similar extents with three different secondary amines. Eighty-eight % of the NSA occurred in the skin, one-third of which was in the hair. The high skin concentration occurred when the bodies but not the heads of mice were exposed to NO2, indicating that the major exposure route was the skin. The NSA might consist of alpha-nitro or other activated nitrite esters derived from unsaturated lipids.