Acridine orange (AO) is the most notable member of the acridine fluorescent dye family. Because of its versatility and diverse biological applications, it has been widely used in basic and clinical research fields (1 –10 ). For flow cytometric application, AO offers distinct advantages over other fluorochromes by simultaneously binding to DNA and RNA in individual cells. RNA quantitation augments conventional DNA analysis by indirectly assessing the biological activity of tumor populations as it relates to the transcriptional activity, cell proliferation, and differentiation status of cells (11 ). However, flow cytometric application of this dye remains limited, because of concerns regarding procedure tediousness and its notoriety for contaminating tubing of flow cytometers. These difficulties can be overcome and successful results can be obtained by adherence to strict staining protocols and careful maintenance of instrumentation.