We studied the influence on circadian rhythms of peptides that have been reported to be colocalized in suprachiasmatic nucleus (SCN) neurons. Gastrin-releasing peptide (GRP1–27), peptide histidine isoleucine (PHI), and vasoactive intestinal polypeptide (VIP) were microinjected into the suprachiasmatic nucleus (SCN) region of Syrian hamsters free running under three different constant lighting conditions. All peptide injections caused phase-dependent phase shifts of hamster locomotor activity rhythms which were unaffected by constant lighting conditions. GRP1–27 (150 pmol) caused large phase delays when injected at circadian times (CT) 12–16, modest phase advances when administered at CT20–24, and few shifts during the subjective day. Injections of saline vehicle at any of these phases caused only very small phase shifts. Phase delays induced by GRP1–27 at CT12–14 were dose dependent, unrelated to injection volume (at a constant dose), and attenuated by pretreatment with the BN/GRP-preferring receptor antagonist BIM 26226. VIP (150 pmol) caused moderate phase delays at CT12–14 and moderate phase advances at CT20–24. PHI (150 pmol) caused moderate phase delays at CT12–14 only. Coadministration of 150 pmol of GRP1–27, PHI, and VIP in an equimolar neuropeptide cocktail (50 pmol of each peptide) caused phase delays at CT12–14 and phase advances at CT20- 24 which did not differ from those induced by 150 pmol of GRP1–27 alone at these phases. The shifts induced by 150 pmol of the peptide cocktail were smaller than the sum of the shifts induced by 50 pmol doses of each peptide administered separately at those phases. Since the phase- delaying effects of the cocktail were weaker than the summed effects of the component 50 pmol doses of the peptides, these data demonstrate a lack of synergism among the effects of these peptides. Since GRP1–27 (150 pmol) evoked shifts similar in magnitude to those of the cocktail, there is no evidence that these apparently colocalized neuropeptides must interact to exert maximal effects on the circadian pacemaker.