The aim of this study was to identify the melatonin receptor type(s) (MT(1) or MT(2)) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[(125)I]iodomelatonin and in situ hybridization histochemistry, with either (33)P- or digoxigenin-labeled antisense MT(1) and MT(2) melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT(1)/MT(2) nonselective luzindole and the MT(2)-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 microM) did not affect circadian phase on their own; however, they blocked both the phase advances (approximately 4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT(2) melatonin receptor signaling.