Ozone toxicity in the lung is thought to be mediated by products derived from the reaction of ozone with components of the lung epithelial lining fluid. Cholesterol is an abundant component of this epithelial lining fluid, and it is susceptible to ozonolysis, yielding several stable products including 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and 5beta,6beta-epoxycholesterol. Both 5beta,6beta-epoxycholesterol and its metabolite, cholestan-6-oxo-3,5-diol, have been shown to cause cytotoxicity in vitro, suggesting that they may be potential mediators of ozone toxicity in vivo. An ozone-sensitive mouse strain, C57BL/6J, was exposed to varying concentrations of ozone (0.5-3.0 ppm), and subsequently the levels of these cholesterol ozonolysis products were quantitated by electrospray ionization mass spectrometry in bronchoalveolar lavage fluid, lavaged cells, and lung homogenate. An ozone dose-dependent formation of these biologically active oxysterols was observed in vivo, supporting a role for these compounds in ozone toxicity. Since the 5beta,6beta-epoxycholesterol metabolite, cholestan-6-oxo-3,5-diol, was isobaric with other cholesterol ozonolysis products, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol condensation product, 3beta-hydroxy-5beta-hydroxy-B-norcholestan-6beta-carboxaldehyde, detailed mass spectral analysis using electron impact ionization was utilized to differentiate these isobaric cholesterol ozonolysis products. The specific detection of cholestan-6-oxo-3,5-diol in lung homogenate after ozone exposure established formation of 5beta,6beta-epoxycholesterol within the lung after exposure to 0.5 ppm ozone.