The utility of the antiproliferative agent fluorouracil has been demonstrated recently in glaucoma-filtering surgery and in the management of proliferative vitreoretinopathy after vitrectomy. Because of the potential for contact with the corneal endothelium, its toxicity to the endothelium is an important consideration. The effect of fluorouracil on corneal endothelial integrity in an in vitro animal model was studied. Freshly excised sheep corneal buttons were exposed to increasing concentrations of fluorouracil for four hours and were subsequently evaluated microscopically using vital staining with alizarin red and trypan blue. Exposure to normal saline and chlorhexidine gluconate was used to establish negative- and positive-staining controls. Corneal buttons exposed to normal saline or to 1.0 mg/mL or less of fluorouracil had intact endothelial monolayers that completely excluded trypan blue. Corneal buttons exposed to chlorhexidine gluconate or to 10 mg/mL of fluorouracil, however, had severely or totally disrupted endothelial monolayers in which the remaining cells, if any, all had blue-staining nuclei. These results suggest that the threshold concentration for fluorouracil toxicity to corneal endothelium lies between 1 and 10 mg/mL when exposure time is four hours.