Nile red, a benzophenoxazine dye, is reported to be an excellent vital stain for detecting intracellular lipid droplets using fluorescence microscopy and flow cytofluorometry. The dye's specificity for lipid droplets was assessed in cultured aortic smooth muscle cells and peritoneal macrophages incubated with acetylated low-density lipoprotein to induce cytoplasmic lipid overloading. Nile red exhibited better selectivity for cytoplasmic lipid droplets when viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, > 528 nm) compared to red fluorescence (excitation, 515-560 nm; emission, > 590 nm). Nile red-stained lipid droplet-filled macrophages showed greater fluorescence intensity than control macrophages, and these differences could be differentiated and analyzed by flow cytofluorometry. Nile red is strongly fluorescent only in the presence of a hydrophobic environment, is highly soluble in lipids, and does not interact with other tissue components except in solution. The dye can be applied to cells in an aqueous medium without dissolving the lipids it is intended to reveal. The study demonstrates the utility of Nile red as a stain for detecting intracellular lipid droplets and its potential for flow cytofluorometric analysis of lipid-laden cells.Nile red, a benzophenoxazine dye, is reported to be an excellent vital stain for detecting intracellular lipid droplets using fluorescence microscopy and flow cytofluorometry. The dye's specificity for lipid droplets was assessed in cultured aortic smooth muscle cells and peritoneal macrophages incubated with acetylated low-density lipoprotein to induce cytoplasmic lipid overloading. Nile red exhibited better selectivity for cytoplasmic lipid droplets when viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, > 528 nm) compared to red fluorescence (excitation, 515-560 nm; emission, > 590 nm). Nile red-stained lipid droplet-filled macrophages showed greater fluorescence intensity than control macrophages, and these differences could be differentiated and analyzed by flow cytofluorometry. Nile red is strongly fluorescent only in the presence of a hydrophobic environment, is highly soluble in lipids, and does not interact with other tissue components except in solution. The dye can be applied to cells in an aqueous medium without dissolving the lipids it is intended to reveal. The study demonstrates the utility of Nile red as a stain for detecting intracellular lipid droplets and its potential for flow cytofluorometric analysis of lipid-laden cells.