The stability of amphotericin B 5 mg/mL in 5% dextrose ophthalmic solution prepared by the Hospital Pharmacy Service was studied in different conditions of storage and use. Admixtures of amphotericin B were aseptically prepared in low-density polyethylene dropper bottles. The stability of amphotericin B was evaluated in ophthalmic dropper bottles stored in a refrigerator, at room temperature, protected from, or exposed to, light. To simulate the effect of exposure to air, some ophthalmic dropper bottles were opened twice daily and two drops were removed. Immediately after preparation, samples were collected to determine the initial drug concentration by high-performance liquid chromatography and to assess pH, osmolality and sterility. The same tests were conducted after four, eight and 15 days of storage in ophthalmic containers opened daily and unopened after eight, 15, 30, 60, 75 and 120 days of storage. Samples were visually inspected daily for signs of physical incompatibility. An additional study was conducted in four ophthalmic containers collected in the ophthalmology unit after eight or 15 days of current patient use testing the same parameters. Ophthamlic containers stored in the refrigerator (the closed and the opened daily set) showed no loss or deterioration of amphotericin B during the corresponding period of storage (120 and 15 days, respectively). We observed precipitation and degradation after 13 days of storage in ophthalmic containers exposed to normal lighting conditions at room temperature, and after 16 days in ophthalmic containers protected from light. There was no appreciable change in pH or osmolality in any of the samples. Microbiological invesigation disclosed negative culture results for all samples. This study shows that aseptically prepared amphotericin B ophthalmic solution packaged in low-density polyethylene bottles can be stored safely for up to 120 days when unopened and stored at 4 deg C and protected from light, for 16 days when stored at 22 deg C and protected from light and for 13 days when stored at 22 deg C and exposed to light.