Cholesterol is the most abundant neutral lipid present in the surfactant of the lung epithelial lining fluid. The double bond between carbons 5 and 6 of cholesterol is susceptible to attack by ozone within this surfactant environment. 6-Oxo-3,5,-diol is a major metabolite of cholesterol formed during exposure of lung epithelial cells to ozone, with formation of 5β,6β-epoxycholesterol as a predominant precursor. Exposure of C57BL/6J mice to 0.5-3 ppm ozone produced a dose-dependent formation of 6-oxo-3,5,-diol which was detectable in the bronchalveolar lavage fluid, lavaged cells, and lung homogenates. 6-Oxo-3,5-diol is a potent inhibitor of cholesterol synthesis in human bronchial epithelial cells with an IC50 of 350 nM and exhibits significant cytotoxicity in the low µM range. Therefore, the toxic effects of ozone may be mediated by formation oxysterols of this type.