Latent inhibition is a phenomenon by which pre-exposure to a conditioned-stimulus (CS), prior to subsequent pairings of that same CS with an unconditioned-stimulus (US), results in decreased conditioned responding to the CS. Previous work in our laboratory has suggested that the entorhinal cortex is critically involved in the establishment of latent inhibition of cued fear conditioning. Furthermore, utilizing systemic pharmacology, we have demonstrated a role for of NMDA receptors, protein kinase A (PKA), and mitogen activated protein kinase (MAPK, also known as ERK) in latent inhibition of cued fear conditioning, but until now, where these cell signaling cascades are critically activated during latent inhibition of cued fear was unknown. Here, we use direct drug infusion to demonstrate that cell signaling via NMDA receptors, the cAMP/PKA pathway, and the MAPK pathway within the entorhinal cortex are critically involved in latent inhibition of cued fear conditioning. In the present study, CS pre-exposed mice received 20 CS pre-exposures 24h prior to two pairings of the same CS with a 0.53 mA foot shock US, while control animals receive no pre-exposure to the CS. The NMDA antagonist APV (0.25 or 2.5 microg/side), the cAMP inhibitor Rp-cAMP (1.8 or 18.0 microg/side), or the MAPK inhibitor U0126 (0.1 or 1.0 microg/side) were directly infused into the entorhinal cortex prior to pre-exposure. All three drugs produced dose-dependent disruptions in latent inhibition of cued fear conditioning. Importantly, none of the drugs had any effect on cued fear conditioning when administered on training day, suggesting that the effects of each of the drugs were specific to CS pre-exposure. These results are discussed in relation to the potential mechanisms of plasticity that support latent inhibition of cued fear conditioning.