We studied the effect of hypoxia on cholesterol accumulation in cultured rabbit aortic smooth muscle cells, which were incubated in a medium with normolipemic rabbit serum (NRS) or hyperlipemic rabbit serum (HRS). The cells were incubated in a humidified atmosphere of either 20% O2, 75% N2 and 5% CO2 (control cells) or 2% O2, 93% N2 and 5% CO2 (hypoxic cells). In a medium containing 20% NRS, the free cholesterol level of hypoxic cells was only a little higher than that of control cells, and there was no significant difference in esterified cholesterol content. On the other hand, in a medium containing 20% HRS, the free cholesterol level was slightly higher and the esterified cholesterol level was markedly higher in hypoxic cells compared with control cells. These results show that hypoxia promotes the accumulation of cholesterol, especially as ester, in smooth muscle cells cultured with hyperlipemic serum. These in vitro experiments indicate that hypoxia in the arterial wall associated with hyperlipidemia may play an important role in atherogenesis, although the precise mechanism remains unclear.