Cholesterol oxidation products or oxysterols are of interest due to their hypothesized role in the development of atherosclerosis. The objective of the present study was to assess the cytotoxic effects of mixtures of oxysterols: 25-hydroxycholesterol (25-OHC), 7 beta-hydroxycholesterol (7 beta-OHC), and cholesterol-5 beta,6 beta-epoxide (beta-epox) on two cell types associated with the atherosclerotic process, bovine aortic endothelial (BAE) cells and human monocytic U937 cells. Cells were exposed to 25-OHC, 7 beta-OHC, or beta-epox, or equimolar mixtures (30 mu M) of 25-OHC and 7 beta-OHC, 25-OHC and beta-epox, or 7 beta-OHC and beta-epox for 48 h. Cell viability was assessed using the fluorescein diacetate/ethidium bromide (FDA/EtBr) assay and nuclear morphology following staining with Hoechst 33342. 25-OHC was the least toxic of the oxysterols and did not induce apoptosis in either cell line. Both 7 beta-OHC and beta-epox treatments were cytotoxic and induced apoptosis in the cells. Cotreatment with 25-OHC did not alter the toxicity of 7 beta-OHC and beta-epox in U937 cells but did decrease the percentage apoptotic cell death. In contrast, in the BAE cells cotreatment with 25-OHC had a slight protective effect on 7 beta-OHC and beta-epox-induced toxicities and a marked decrease in apoptotic cell death. The 7 beta-OHC and beta-epox mixture induced a significant increase in apoptotic cell death in U937 cells but decreased this mode of cell death in the BAE cells. The effects of oxysterols on glutathione levels also differed between the cells with changes noted in U937 and not in BAE cells. Results demonstrate interactive effects when oxysterols are studied as mixtures rather than single compounds in vitro.