Cholesterol oxidation products (oxysterols) are generated during the cooking and processing of foods and may be produced endogenously in tissues and in the plasma membrane. A diverse range of biological functions have been ascribed to oxysterols, including atherogenicity, carcinogenicity, and mutagenicity, and in recent years concern has been expressed over the presence of oxysterols in food products. However, it is unclear whether oxysterols are capable of inducing genotoxic damage in cell culture systems. The aim of this study was to examine seven commonly occurring oxysterols (purity > 95%) for their cytotoxicity and ability to increase the frequency of DNA strand breaks and sister chromatid exchanges (SCE) in cells in culture. Two cell lines were employed in the study: Chinese hamster ovary (CHO) and Indian Muntjac (IM) fibroblasts. The 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay, which is a measure of intracellular reductive metabolism based on the activity of mitochondrial dehydrogenases, was used as an index of cytotoxicity. The most cytotoxic oxysterols in constantly challenged CHO or IM cells (24-h exposure) proved to be 5 alpha-cholestane-3 beta,5,6 beta-triol and 25-hydroxy-cholesterol. The genotoxic potential of the oxysterols was assessed in CHO cells using the comet assay and IM cells using the SCE assay. The comet assay measures breaks in the DNA strand, whereas the exact mechanism of SCE formation is unclear but is believed to require DNA repair where genetic material becomes exchanged between the two sister chromatids. None of the oxysterols examined in this study affected baseline levels of DNA strand breaks or SCE relative to the negative control samples. This study indicates that, under the conditions used, the oxysterols investigated were not genotoxic.