Nile red (9-diethylamino-5H-benzo[alpha]phenoxazine-5-one) is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. It has environment-sensitive fluorescence. Nile red is intensely fluorescent in a lipid-rich environment while it has minimal fluorescence in aqueous media. Better selectivity for cytoplasmic lipid droplets can be obtained when the cells are viewed with yellow-gold fluorescence (450-500 nm excitation; >528 nm emission) rather than red fluorescence (515-560 nm excitation; >590 nm emission). Nile red is strongly fluorescent, but only in a hydrophobic environment.