Acetylcholine (ACh) was the first neurotransmitter identified as a regulator of mammalian circadian rhythms. When injected in vivo, cholinergics induced biphasic clock resetting at night, similar to nocturnal light exposure. However, the retinohypothalamic tract connecting the eye to the suprachiasmatic nucleus (SCN) uses glutamate (GLU) to transmit light signals. We here resolve this long-standing paradox. Whereas injection of the cholinergic agonist, carbachol, into the mouse ventricular system in vivo induced light-like effects, direct application to the SCN in vitro or in vivo induced a distinct response pattern: phase advance of circadian rhythms throughout the nighttime. These results indicate that a new regulatory pathway, involving an extra-SCN cholinergic synapse accessible via ventricular injection, mediates the light-like cholinergic clock resetting reported previously.