Melatonin solutions are frequently used in human, animal, and in vitro research. Generally, fresh solutions are prepared, for fear of instability of melatonin in solution. We tested the high-performance liquid chromatography (HPLC) stability of melatonin in aqueous solutions stored room temperature, 4 degrees C, and -70 degrees C for up to 6 months. Solutions were prepared in a laminar flow hood using sterile technique, directly into sterile, pyrogen-free glass vacuum vials for storage. Different concentrations were tested (1.0-113.0 micrograms/ml). There was no loss of potency as assessed by HPLC, and the preparations remained sterile and pyrogen-free. We conclude that melatonin solutions may be prepared in batches maintained in sterile, pyrogen-free vials at 4 degrees C or at -70 degrees C until use within 6 months. This method will save on research time used for preparation of fresh solutions and will reduce the number of dose validation tests for each new experiment.