Mammalian circadian rhythms are controlled by the suprachiasmatic nuclei (SCN) in concert with light information. Several neurotransmitters and neural pathways modulate light effects on SCN timing. This study used a line of rat with an upregulated cholinergic system to investigate the role of acetylcholine in rhythmicity. With the use of a selective breeding program based on the thermic response to a cholinergic agonist, we developed a supersensitive (S_ox) and subsensitive (R_ox) rat line. The S_ox rats showed an earlier onset time of melatonin rhythm under a 12:12-h light-dark photoperiod from generation 3 (3 ± 0.5 h after dark) compared with R_ox rats (4.5 ± 0.1 h) and an earlier morning decline in temperature (0.9 ± 0.3 h before lights on) compared with R_ox animals (0.1 ± 0.1 h). Furthermore, the S_oxanimals displayed a significantly shorter free-running period of temperature rhythm than R_ox rats (23.9 ± 0.04 and 24.3 ± 0.1 h, respectively, P < 0.05). The results suggest that the altered circadian timing of the S_ox rats may be related to the cholinergic supersensitivity, intimating a role for acetylcholine in the circadian timing system.