Metabolically active cells are able to convert the MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye to blue formazan. This is the basis of the MTT assay, which is among the most widely used screening methods to evaluate cell viability and proliferation. When testing the effects of cholesterol products on the viability of human pulmonary epithelial-like A549 cells using trypan blue staining (cell numbers) and the MTT assay, results were inconsistent. The MTT assay indicated greater than 50% loss of viability with exposure of cells to cholesterol, whereas there was no decrease in viability indicated by trypan blue exclusion and propidium iodide uptake. A similar decrease in MTT reduction was obtained upon cholesterol treatment in human lung microvascular endothelial cells (HLMVECs) and human coronary artery endothelial cells (HCAECs) without loss of viability. This suggested a direct interference of cholesterol with the assay. However, using a cell-free system, there was no decrease in the reduction of MTT by ascorbic acid during incubation with a similar concentration of cholesterol. Light microscopy revealed enhanced exocytosis of formazan granules in presence of cholesterol. Incubation with apolipoprotein A-1 decreased cholesterol-mediated inhibition of MTT assay. These studies indicate decreased MTT reduction as a result of enhanced exocytosis of formazan due to cholesterol. A careful validation of viability assay procedures is therefore suggested in experiments where cholesterol is a constituent, to avoid a potential bias in concluding results of cytotoxicity studies.