Monolayers of L-cell cultures were incubated with oxygenated cholesterols and cellular protein was subsequently measured to assess the cytotoxicity of the cholesterol oxides. The site of oxidation of the cholesterol molecule was not reflected in the degree of cytotoxicity. Of the 12 cholesterol oxides tested, cholestan-3 beta, 5 alpha, 6 beta-triol and cholestan-3,5-diene-7-one exhibited the greatest cytotoxicity, approximately 45% inhibition (reduction) in protein compared to the control at concentrations of 10 micrograms/ml, after 1 day of incubation with the compounds. 5-Cholesten-3 beta, 25-diol and 5-cholesten-3 beta-ol-7-one were the most cytotoxic after 5 days' exposure with concentrations of 10 micrograms/ml reducing the protein content to approximately 10% of the control. Lipid accumulation in the L cells (steatosis), as measured by Oil Red O-stainable droplets formed in response to cholesterol oxides was also investigated. Grade 3 steatosis (an increase in lipid droplets so that more than 75% of the L-cell cytoplasm was occupied by lipid droplets) after 1 day of exposure to cholesterol oxides generally reflected the cytotoxic effect of the same cholesterol oxides after 5 days' exposure. However some of the steatotic cholesterol oxides, such as the alpha and beta epimers of cholestan-5,6-epoxy-3 beta-ol were highly active with respect to steatosis, increasing the grade 3 value to 209 and 390% of control, respectively, but were not cytotoxic. This has led to the suggestion that the cholesterol oxides have more than one mode of action in L cells.