The brain's biological clock, which, in mammals, is located in the suprachiasmatic nucleus (SCN), generates circadian rhythms in behaviour and physiology. These biological rhythms are adjusted daily (entrained) to the environmental light/dark cycle via a monosynaptic retinofugal pathway, the retinohypothalamic tract (RHT). In this review, the anatomical and physiological evidence for glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as principal transmitters of the RHT will be considered. A combination of immunohistochemistry at both the light- and electron-microscopic levels and tract-tracing studies have revealed that these two transmitters are co-stored in a subpopulation of retinal ganglion cells projecting to the retino-recipient zone of the ventral SCN. The PACAP/glutamate-containing cells, which constitute the RHT, also contain a recently identified photoreceptor protein, melanopsin, which may function as a "circadian photopigment". In vivo and in vitro studies have shown that glutamate and glutamate agonists such as N-methyl- D-aspartate mimic light-induced phase shifts and that application of glutamate antagonists blocks light-induced phase shifts at subjective night indicating that glutamate mediates light signalling to the clock. PACAP in nanomolar concentrations has similar phase-shifting capacity as light and glutamate, whereas PACAP in micromolar concentrations modulates glutamate-induced phase shifts. Possible targets for PACAP and glutamate are the recently identified clock genes Per1 and Per2, which are induced in the SCN by light, glutamate and PACAP at night.