This paper describes the inhibitory activities of cholesterol derivatives such as cholesterol, sodium cholesteryl sulfate, cholesteryl-5alpha, 6alpha-epoxide, cholesteryl chloride, cholesteryl bromide, and cholesteryl hemisuccinate (compounds 1-6, respectively) against DNA polymerase (pol), DNA topoisomerase (topo), and human cancer cell growth. Among the compounds tested, compounds 2 and 6 revealed themselves to be potent inhibitors of animal pols, and the IC50 values for pols were 0.84-11.6 and 2.9-148 microM, respectively. Compounds 2, 3 and 6 inhibited the activity of human topo II, with IC50 values of 5.0, 12.5 and 120 microM, respectively. Compounds 2, 3 and 6 also suppressed human cancer cell (promyelocytic leukemia cell line, HL-60) growth, and LD50 values were 8.8, 20.2 and 72.3 microM, respectively, suggesting that cell growth inhibition had the same tendency as the inhibition of topos rather than pols. Compounds 2 and 6 arrested the cells in S and G2/M phases, compound 3 arrested the cells in the G2/M phase, and these compounds also increased sub-G1 phase in the cell cycle. These results suggested that the effect of cell cycle arrest might be effective on both pols and topos activities. From these findings, the action mode of cholesterol derivatives as anti-cancer compounds is discussed.