5-Fluorouracil (5-FU) and Mitomycin C (MMC) are used as adjunct chemotherapy during glaucoma filtering surgery to suppress conjunctival fibroblast proliferation. Since part of these agents may gain access to the anterior chamber and cause cytotoxicity to the corneal endothelium we set up an in vitro system to establish a dose-response effect. Cytotoxicity of MMC and 5-FU was quantified using Mosmann's colorimetric assay in a bovine endothelial cell culture system. In this assay the respiratory activity of the cells is used as a marker for cell viability.After incubation for 5 minutes the 3.0 mg/ml concentration of MMC showed endothelial cytotoxicity, whereas no endothelial toxicity of 5-FU was noted in concentrations up to 50 mg/ml. After incubation for 30 minutes endothelial cytotoxicity was demonstrated for 50 mg/ml of 5-FU and 1 mg/ml of MMC. After an exposure-time of 60 minutes the toxicity level remained 50 mg/ml for 5-FU but decreased to 0.5 mg/ml for MMC.We conclude that with respect to the clinically used concentrations and methods of application of 5-FU and MMC in vivo endothelial toxicity is not to be expected. However, in cases of accidental access of MMC to the anterior eye chamber and following a reduction of aqueous turnover rate the safety of MMC is unwarranted.