We investigated the combined fluorescent dyes acridine orange (AO) and ethidium bromide (EB) for corneal endothelial evaluation. Both dyes intercalate with DNA and RNA and are mutagenic at high concentrations. Optimum differential staining was obtained after 5 minutes exposure to 1 microgram/ml of each dye. Electron microscopy confirmed that the dye combination simultaneously identified both viable and nonviable cells. Swelling of perfused dog corneas for 3 hours after exposure to both dyes for 5 minutes was not increased. Exposure to visible light from a fluorescence microscope for 2-5 minutes caused increased corneal swelling and abnormal intercellular junctions by electron microscopy in paired dog corneas stained for 5 minutes with either AO or EB. The addition of 10% calf serum to the staining and observation media decreased the toxic effect of AO/EB and light seen by specular microscopy and by electron microscopy. Human corneas exposed to AO and EB were not mutagenic in the Salmonella/microsome assay. These results indicate that AO and EB, 1 microgram/ml, provide a rapid identification of both viable and nonviable corneal endothelial cells. When the corneas are exposed to light, however, the dyes are toxic; this toxicity is decreased by the presence of 10% calf serum in the staining and observation media.