Persistent neuronal activity lasting seconds to minutes has been proposed to allow for the transient storage of memory traces in entorhinal cortex and thus could play a major role in working memory. Nonsynaptic plateau potentials induced by acetylcholine account for persistent firing in many cortical and subcortical structures. The expression of these intrinsic properties in cortical neurons involves the recruitment of a non-selective cation conductance. Despite its functional importance, the identity of the cation channels remains unknown. Here we show that, in layer V of rat medial entorhinal cortex, muscarinic receptor-evoked plateau potentials and persistent firing induced by carbachol require phospholipase C activation, decrease of PIP(2) levels, and permissive intracellular Ca(2+) concentrations. Plateau potentials and persistent activity were suppressed by the generic nonselective cation channel blockers FFA (100 μM) and 2-APB (100 μM), as well as by the TRPC channel blocker SKF-96365 (50 μM). However, plateau potentials were not affected by the TRPV channel blocker ruthenium red (40 μM). The TRPC3/6/7 activator OAG did not induce or enhance persistent firing evoked by carbachol. Voltage clamp recordings revealed a carbachol-activated, nonselective cationic current with a heteromeric TRPC-like phenotype. Moreover, plateau potentials and persistent firing were inhibited by intracellular application of the peptide EQVTTRL that disrupts interactions between the C-terminal domain of TRPC4/5 subunits and associated PDZ proteins. Altogether, our data suggest that TRPC cation channels mediating persistent muscarinic currents significantly contribute to the firing and mnemonic properties of projection neurons in the entorhinal cortex.