A significant accumulation of cellular free cholesterol and steryl esters is observed in J774 macrophages when cells are exposed to low-density lipoproteins (LDL) containing cholesterol 5β,6β-epoxide. This cellular sterol accumulation is mainly due to the formation of esterified cholesterol and desmosterol. Cellular steryl esters increased to 39.4 and 22.4 μg/mg cell protein with 0.8 μM of cholesterol 5β,6β-epoxide and 3,5-cholestadien-7-one, respectively, whereas hardly detectable levels were observed with the absence of oxysterols. The total cellular sterols increased 45% above the value of control with cholesterol 5β,6β-epoxide. The uptake of [³H] cholesteryl oleate-LDL was also enhanced by cholesterol 5β,6β-epoxide. The rapid displacement of desmosterol with cholesterol was observed when cells were treated with cholesterol 5β,6β-epoxide or 3,5-cholestadien-7-one in the presence of LDL. Cholesterol 5β,6β-epoxide became associated with LDL in the culture conditions, and its uptake into J774 cells and the cytotoxicity were reduced significantly by the association with LDL. The comparison of selected oxysterols for their ability to stimulate cellular sterol accumulation indicated that cholesterol 5β,6β-epoxide is the most potent. Cholesterol esterification was enhanced significantly by cholesterol 5β,6β-epoxide whereas cholesterol 5α,6α-epoxide and 3,5-cholestadien-7-one produced a modest response. In contrast, although cholestantriol, the metabolic hydrolysis product of cholesterol epoxides, also associated with LDL, it showed no stimulating effect on both cellular sterol content and sterol esterification. These results indicate that some oxysterols, such as cholesterol 5β,6β-epoxide and possibly 3,5-cholestadien-7-one, stimulate cellular sterol accumulation in J774 macrophages and may play an important role in atherogenesis.