The effect of monensin on the cytotoxic effect of antitransferrin receptor immunotoxin (IT) was determined on cultured, human retinal pigment epithelial (hRPE) cells. Human RPE cells were treated with 0.1 - 10,000 ng/ml IT with and without 0.01 - 0.1μM monensin, a lysosomotropic reagent that can influence IT activity. Monensin (0.01μM) shortened the onset of cell kill with IT (10,000 ng/ml) from 48 to 24 hours (p = 0.0016). Although 0.01μM monensin alone was not cytotoxic to hRPE cells, a single 7 - day treatment with monensin caused up to a 4.1 - fold increase in antiproliferative potency of IT on proliferating hRPE cells (p≤0.0001). Enhancement was obtained with only a 1 - hour exposure to 0.1μM monensin (p = 0.0001). In contrast, IT (0.1 - 10,000 ng/ml) combined with monensin (0.01μM) had minimal effect on density - arrested cells. IT with or without monensin did not inhibit proliferation of Rhesus monkey RPE cells. Our results indicate that monensin enhances the selective cytotoxic effect of IT on proliferating hRPE cells in culture.