We investigated the effects of 0.5, 2.5, and 10 micrograms/ml of cholesterol or 26-hydroxycholesterol on bovine aortic ECs and SMCs. Suppression of viable cell density and cytotoxic changes in both cells were induced by 2.5 and 10 micrograms/ml 26-hydroxycholesterol. ECs were more severely damaged than SMCs in the presence of 26-hydroxycholesterol. Levels of up to 10 micrograms/ml cholesterol had no effect on ECs or SMCs growth or cytotoxicity. Confluent ECs exposed to 2.5 or 10 micrograms/ml of 26-hydroxycholesterol secreted significant amounts of 6-ketoprostaglandin F1 alpha after a 24-hr incubation. An equivalent concentration of cholesterol had no such effect. SMCs cocultured with ECs exposed to 10 micrograms/ml 26-hydroxycholesterol, when compared with an equivalent level of cholesterol, synthesized DNA in significantly greater amounts during a 24-hr incubation. The cocultured ECs incubated in the presence of 2.5 and 10 micrograms/ml 26-hydroxycholesterol were partially detached due to cell death. However, no difference was observed in the DNA content of SMCs cultured without ECs in the presence of cholesterol or 26-hydroxycholesterol. The results suggest that 26-hydroxycholesterol produced not only cytotoxicity to ECs and SMCs but also stimulated DNA synthesis in SMCs through endothelial injury.