Intact cholinergic innervation of visual cortex is critical for normal processing of visual information and for spatial memory acquisition and retention. However, a complete description of the mechanisms by which the cholinergic system modifies synaptic function in visual cortex is lacking. Previously it was shown that activation of the m1 subtype of muscarinic receptor induces an activity-dependent and partially N-methyl-D-aspartate receptor (NMDAR)-dependent long-term depression (LTD) at layer 4-layer 2/3 synapses in rat visual cortex slices in vitro. The cellular mechanisms downstream of the Gαq coupled m1 receptor required for induction of this LTD (which we term mLTD) are currently unknown. Here, we confirm a role for m1 receptors in mLTD induction and use a series of pharmacological tools to study the signaling molecules downstream of m1 receptor activation in mLTD induction. We found that mLTD is prevented by inhibitors of L-type Ca2+ channels, the Src kinase family, and the mitogen-activated kinase/extracellular kinase. mLTD is also partially dependent on phospholipase C but is unaffected by blocking protein kinase C. mLTD expression can be long-lasting (>2 h) and its long-term maintenance requires translation. Thus we report the signaling mechanisms underlying induction of an m1 receptor-dependent LTD in visual cortex and the requirement of protein synthesis for long-term expression. This plasticity could be a mechanism by which the cholinergic system modifies glutamatergic synapse function to permit normal visual system processing required for cognition. Copyright © 2007 The American Physiological Society.