The aim of the research reported here was to provide information on the neurochemical processes that underlie the generation and entrainment of mammalian circadian rhythms. The studies were centered principally around the in vivo brain microdialysis technique for assessing the daily pattern of neurotransmitter activity in the suprachiasmatic hypothalamus of freely behaving Syrian and Siberian hamsters. This approach yielded several findings related to the activities of serotonergic and excitatory amino acid systems in the region of the suprachiasmatic nuclei (SNC). Specifically, we found that (1) there were daily variations in the extracellular concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and glutamate (GLU) in the SCN region (highest levels of 5-HIAA occurred soon after lights-off, whereas GLU peaked later in the dark phase); (2) the daily rhythm of GLU, but not serotonin, in the SCN region appeared to be circadian in nature and was not driven by an external influence; (3) the rhythm in GLU measured in SCN microdialysate involved a tetrodotoxin-insensitive mechanism and did not appear to be directly linked to the expression of locomotor behavior; and (4) direct application of serotonin receptor agonists via the microdialysis probe suppressed the concentration of extracellular GLU in the SCN region, suggesting that serotonin may modulate GLU release in the SCN.