Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of several oxysterols to induce short-term death in cancerous (human breast cancer and mouse skin melanoma cells) and non-cancerous (human endothelial cells and lung fibroblasts) cell lines. We determined cell viability, Ki67 expression, cell cycle regulation, and apoptosis after 24-h incubations with oxysterols. We found that different oxysterols had different effects on the studied parameters. Moreover, the effects depended on cell type and oxysterol concentration. Three cytotoxic oxysterols (7-ketocholesterol, cholestane-3 beta-5 alpha-6 beta-triol, and 5 alpha-cholestane-3 beta,6 beta-diol) inhibited the S phase and stimulated the G0/G1 or G2/M phases. These oxysterols promoted apoptosis, determined with Annexin V and propidium iodide assays. These results showed that different oxysterols have cytotoxic effects depending on the cell line. The findings suggest a potential pharmacological utility of cytotoxic oxysterols. (C) 2018 Elsevier Inc. All rights reserved.