The mechanisms which mediate non-photic entrainment of the circadian clock are not known. The free-running circadian rhythm of the wheel-running activity of Syrian hamsters was phase advanced by subcutaneous injection of saline and the associated handling at circadian time (CT) 10 (2 h before activity onset, CT 12). Over several experiments, approximately 80% of the animals were sensitive to this non-photic cue and were, therefore, designated as 'shifters'. Injection at CT 6 had no phase-shifting effect in any animals. A series of six injections presented to shifters at CT 10 over 14 circadian cycles produced a cumulative phase advance to the activity rhythm which free ran from the phase established by the final injection, demonstrating that the non-photic stimulus caused true entrainment of the circadian clock, rather than simply masking its expression. Entrainment by light is associated with the rapid phosphorylation of the transcription regulatory factor, cyclic adenosine monophosphate response element binding protein (CREB), in the suprachiasmatic nuclei (SCN), the principal circadian clock. The induction of immunoreactivity to the phosphorylated form of CREB (P-CREB-ir) was used to determine whether phosphorylation is also activated by a non-photic phase-shifting cue. No significant P-CREB-ir was found in shifters maintained under constant dim red light and sampled at either CT 10.05 or 14.05, i.e., times when animals are sensitive to non-photic stimuli and light pulses, respectively. Exposure of animals to light for 5 min at CT 14 caused a dramatic increase in P-CREB-ir within the SCN at CT 14.05. However, despite being sensitive to the phase-shifting effects of handling and injection at CT 10, none of the shifters sampled 5 min after injection of saline at CT 10 exhibited P-CREB-ir within the SCN. These data demonstrate that non-photic cues do not activate a cellular pathway (phosphorylation of CREB) known to be activated within the SCN by light. To assess the importance of arousal in non-photic entrainment, the effect of injection of saline upon activation of the adrenocortical axis, a peripheral index of arousal, was investigated. As anticipated, there was a marked circadian variation in serum cortisol concentrations. In animals identified as shifters, the levels were low during mid-subjective day (CT 6) and rose in advance of activity onset (CT 10). Injection and handling at CT 6 did not have a did not have a significant effect upon serum cortisol levels. In contrast, injection of shifters at CT 10 was associated with a pronounced rise in serum cortisol levels.(ABSTRACT TRUNCATED AT 400 WORDS)