The effect of melatonin on [3H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 nM) significantly increased [3H]glutamate uptake, and this effect persisted in a Ca2+-free medium. On the other hand, melatonin significantly increased [3H]glutamate release in retinas excised at 2400 h, but this effect was Ca2+ sensitive. Melatonin significantly increased 45Ca2+ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [3H]glutamate uptake, [3H]glutamate release, or 45Ca2+ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8-bromoguanosine 3',5'-cyclic monophosphate and 2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate, significantly increased [3H]-glutamate uptake, [3H]glutamate release, and 45Ca2+ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.