The suprachiasmatic nucleus (SCN) is engaged in modulation of memory retention after (fear) conditioning, but it is unknown which pathways and neurotransmitter system(s) play a role in this action. Here we examine immunocytochemically whether muscarinic acetylcholine receptors (mAChRs), mediating cholinergic signal transduction in the SCN, are involved. For this purpose, mAChR immunoreactivity (mAChR-ir) was studied in the SCN after various stages of passive shock avoidance (PSA) and active shock avoidance (ASA) training and, for ASA, at various posttraining time points. mAChR-ir was significantly enhanced in SCN neurons as a result of the training procedure, and the number of mAChR-positive glial cells in the SCN increased significantly. The increase in mAChR-ir as a result of PSA and ASA training was not due to fear conditioning or the number of correct avoidances (in case of ASA training) but rather to behavioral arousal as a consequence of (brief) exposure to a novel environment (the test apparatus). This finding was confirmed by a cage-change experiment in which the rats were allowed to stay in a novel cage for 15 min or 24 hr. Only the brief exposure to the fresh cage triggered alterations for SCN mAChRs 24 hr later. These results shed new light on a possible function of the cholinergic system in the SCN mediated by mAChRs in relation to modulation of memory processes and demonstrate that behavioral arousal during (the habituation stage of) a learning task is sufficient to alter the mAChR system in the SCN.