Nile Red (9-diethylamino-5H-benzo [alpha] phenoxazine-5-one) is a fluorescent lipophilic dye characterized by a shift of emission from red to yellow according to the degree of hydrophobicity of lipids. Polar lipids (i.e., phospholipids) which are mostly present in membranes, are stained in red whereas neutral lipids (esterified cholesterol and triglycerides) which are present in lipid droplets, are stained in yellow. Besides this marked, qualitative contrast between polar and neutral lipids, small differences of the hydrophobic strength could be assessed by the quantitative ratio of red and yellow emissions, in order to extend the discrimination of lipids within the groups of neutral and polar lipids. On the other hand, ratiometric data of red and yellow emissions have not yet been evaluated in the numerous previous light microscopy investigations which used Nile Red. In this work we show that the Nile Red red/yellow ratio enables discrimination of different lipids (monooleine>oleic acid>phosphatidylcholine>free cholesterol>trioleine>oleyl cholesteryl ester). We also show changes in the Nile Red red/yellow emission ratio of lipid droplets of 3T3 mouse fibroblasts induced by drugs interfering with the cholesterol cycle.