Previous studies have established that microinjection of gastrin-releasing peptide (GRP) into the suprachiasmatic nucleus (SCN) region or third ventricle causes circadian phase shifts similar to those produced by light pulses. Activation of N-methyl-d-aspartate (NMDA) receptors in the SCN region also produces light-like phase shifts. This study was designed to test the effects of (+/-)-2-amino-5-phosphonopentanoic acid (AP5), an NMDA antagonist, and l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a glutamate reuptake inhibitor, on GRP-induced phase shifts. Adult male Syrian hamsters equipped with a surgically implanted guide cannula aimed at the third ventricle were housed in constant darkness until stable free-running rhythms of wheel-running activity were apparent. Microinjection of GRP into the third ventricle at circadian time (CT)13 induced large phase delays. These GRP-induced phase delays were completely blocked by co-administration of AP5, suggesting that GRP-induced phase delays require concurrent activation of NMDA receptors. Microinjection of AP5 alone did not induce significant phase shifts. A second set of experiments was designed to test whether GRP-induced phase shifts would be enhanced by PDC. Co-administration of PDC and GRP elicited significantly larger phase delays at CT13 than GRP alone. However, administration of PDC alone did not induce a significant phase shift. Finally, when administered just prior to a light pulse, PDC elicited significantly larger phase delays than light pulse plus vehicle controls. These data suggest that the effects of GRP on the circadian clock phase are highly dependent on the level of excitation provided by activated NMDA receptors.