Etoposide (VP16), a clinically available anticancer compound known as a potent inhibitor of topoisomerase 11, was demonstrated to inhibit irreversibly cytomegalovirus (CMV) replication. This study was performed to evaluate nontoxic concentrations of etoposide for intravitreal injection and vitrectomy infusion in patients with CMV retinitis. We used the isolated perfused vertebrate retina technique, an electrophysiological in vitro technique, for examination of retinal toxicity in higher vertebrates. Bovine and human retina preparations were perfused with an oxygen-preequilibrated standard solution. The electroretinogram (ERG) was recorded as transretinal potential using AgAgCl electrodes. The ERG serves as an indicator of the integrity of the photoreceptors, the first retinal synapse, and higher retinal neurons. After recording of stable ERG amplitudes, the drug was added to the solution for 45 min in the following concentrations: 0.0063, 0.021,0.063,0.21, mMol/L. The preparation was then reperfused with standard solution for another 45 min. The percentage of b-wave reduction after exposure was calculated. Toxic effects of etoposide were found for concentrations of 0.063 mMol/L (37.5 pg/ml) or higher. The effects after application of the higher concentrations were not reversible within the recovery time. Examination of a few isolated human retina preparations showed similar results. The ERG reflecting first- and higher-order neuron activity of the retina showed toxic effects only at concentrations distinctly exceeding the etoposide concentration found to inhibit irreversibly CMV replication in vitro (2.5 pg/ml). Intravitreal injections of etoposide with due consideration of the therapeutic index may provide a safe and effective means of treatment for patients with CMV retinitis resistant to ganciclovir, foscarnet, and cidofovir.