Acetylcholine (ACh) is a neurotransmitter that plays a variety of roles in the central nervous system (CNS). It was previously shown that blocking muscarinic receptors with a non-selective antagonist prevents a form of experience-dependent plasticity, termed spatiotemporal sequence learning, in the mouse primary visual cortex (V1). Muscarinic signaling is a complex process involving the combined activities of five different G-protein coupled receptors, M1-M5, all of which are expressed in the murine brain but differ from each other functionally and in anatomical localization. Here we present electrophysiological evidence that M2, but not M1, receptors are required for spatiotemporal sequence learning in mouse V1. We show that M2 is highly expressed in the neuropil in V1, especially in thalamorecipient layer 4, and co-localizes to the soma in a subset of somatostatin expressing neurons in deep layers. We also show that expression of M2 receptors is higher in the monocular region of V1 than it is in the binocular region, but that the amount of experience-dependent sequence potentiation is similar in both regions. This work establishes a new functional role for M2-type receptors in processing temporal information and demonstrates that monocular circuits are modified by experience in a manner similar to binocular circuits.