The effects of various oxysterols (7 beta-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5 alpha, 6 alpha-epoxide, and 25-hydroxycholesterol) and of cholesterol were investigated on cell growth of bovine aortic endothelial (BAE) cells by cell counting, MTT reduction, and 3H-thymidine incorporation in a 5 to 80 micrograms/ml concentration range. By cell counting, a dose related decrease in the number of adherent cells was observed with oxysterols; MTT reduction also indicated a decreased number of viable cells, and both method give similar IC50. A lower 3H-thymidine incorporation was generally detected with oxysterols but no effect on 3H-thymidine incorporation was found with 25-hydroxycholesterol. With cholesterol, no modification of cell growth was shown by cell counting and 3H-thymidine incorporation, whereas an important decrease in MTT reduction was observed. Noteworthy, with the highest cholesterol concentration no change in cellular morphology occurred, and no modification of mitochondrial activity was found with Rhodamine 123. It is concluded that MTT and 3H-thymidine incorporation are not suitable for the evaluation of a putative toxicity of cholesterol and 25-hydroxycholesterol, respectively. Therefore, cell counting seems the most accurate method to determine the effects of oxysterols and of cholesterol on endothelial cell growth.