OBJECTIVE To investigate the ultrastructural and physiologic effects of exposure of the human corneal endothelium to mitomycin at concentrations of 20 micrograms/mL and 200 micrograms/mL using electron microscopy and in vitro specular perfusion techniques. METHODS Four pairs of corneas (with one cornea of each pair receiving balanced salt solution [BSS Plus, Alcon Laboratories, Fort Worth, Tex] and the other receiving BSS Plus with 20 micrograms/mL of mitomycin) suitable for transplantation, except for extremes of age or systemic disease, underwent perfusion with corneal thickness measured serially every 15 minutes followed by fixation for electron microscopy. Mean corneal swelling rate was calculated for all four experiments, and the control group that received BSS Plus was compared with the group that received mitomycin using a paired t test. Electron micrographs were examined in a masked fashion. Similar studies were performed using two pairs of corneas that received 200 micrograms/mL of mitomycin. RESULTS The mean swelling rate for corneas perfused with 20 micrograms/mL of mitomycin (-4.1 microns/h) was not significantly different from that seen in tissue perfused with BSS Plus (-4.2 microns/h). No consistent ultrastructural changes could be attributed to exposure to 20 micrograms/mL of mitomycin. Perfusions of mitomycin at 200 micrograms/mL resulted in prompt corneal swelling with marked ultrastructural alterations compared with tissue perfused with BSS Plus. CONCLUSIONS Human corneal endothelium may be exposed to undiluted (200 to 500 micrograms/mL) mitomycin with inadvertent entry into the anterior chamber during dissection of the scleral flap bed in trabeculectomy followed by application of mitomycin. This will result in prompt destruction of the endothelium. Exposure to 20 micrograms/mL of mitomycin, a level exceeding the concentration that may be present in the aqueous humor after its proper application, appears nontoxic in this system.