The present results indicate that 30μg/ml copper was toxic to bovine corneal endothelial cells (BCEC) cultured in a serum-free medium (SFM) when the duration of treatment was 72 hours or more. Copper at 10μg/ml, if mixed with 50μg/ml ascorbate 2 to 3 hours before treatment, caused a transient decrease in the number of nuclei/mm2at 72 hour, whereas 10μg/ml copper alone was apparently non-toxic. When 10μg/ml copper was added to 50μg/ml ascorbate at the time of treatment, the toxicity was increased. All the treated cells failed to survive beyond 24 hours, and copper at a lower concentration of 1μg/ml could inhibit the proliferation of BCEC. We propose that copper toxicity on BCEC is augmented by ascorbate possibly through the increased replenishment of Cu+and the subsequent enhanced production of free radicals by copper auto-oxidation.