Preincubation of synaptosome-rich homogenates of rat hypothalamus with melatonin resulted in significant decreases of norepinephrine, serotonin, dopamine and glutamate uptake. Melatonin inhibition was noncompetitive; apparent Km's of initial uptake processes were: (2.5 +/- 0.3) x 10(-7) M for norepinephrine, (2.6 +/- 0.3) x 10(-7) M for serotonin, (2.4 +/- 0.4) x 10(-7) M for dopamine and (1.0 +/- 0.3) x 10(-7) M for glutamate. Apparent Ki's for melatonin inhibition of transmitter uptake were: 0.64 +/- 0.14 mM (norepinephrine), 0.23 +/- mM (serotonin), 0.51 +/- 0.08 mM (dopamine) and 1.21 +/- 0.10 mM (glutamate). Transmitter release evoked by increasing [K+] in medium to 30 mM was augmented by melatonin in a dose-dependent manner. Maximal effects were observed on serotonin release. Accumulation of 3H-melatonin within synaptosome-rich homogenates did not exhibit differences between 0 and 37 degrees C, indicating that the uptake of the hormone was not an active process. These results suggest that exogenously-administered melatonin may affect neurotransmitter accumulation and release in the hypothalamus by modification of the transmitter uptake mechanism rather than by competition with the transmitter for its uptake pump.