The authors investigated whether fluorescein sodium affects the in vitro endothelial function of rabbit corneas. As an index of this function, the transendothelial electrical potential difference (TEPD) was used. The TEPD in a balanced salts and glucose (BSG) control solution increased for the first 30 min and then decayed slowly, reaching about 60% of its original value after 5 hr. When a BSG solution containing 5 micrograms/ml of fluorescein sodium was used, the TEPD time course was similar to the control solution. Since this fluorescein sodium concentration is about sevenfold higher than that seen in the anterior chamber of ocular patients, these results reassure users that no toxic effect of fluorescein is discernible at concentrations relevant to ophthalmic practice. With a fluorescein sodium concentration of 500 micrograms/ml, the TEPD decreased below control values after 4 hr of exposure, but such a concentration is approximately 5000-fold higher than that seen in the anterior chamber of patients. The adverse effect of fluorescein on TEPD is probably irrelevant for standard systemic clinical use.