Trace conditioning is considered a model of higher cognitive involvement in simple associative tasks. Studies of trace conditioning have shown that cortical areas and the hippocampal formation are required to associate events that occur at different times. However, the mechanisms that bridge the trace interval during the acquisition of trace conditioning remain unknown. In four experiments with fear conditioning in rats, we explored the involvement of the entorhinal cortex (EC) in the acquisition of fear under a trace-30 s protocol. We first determined that pretraining neurotoxic lesions of the EC selectively impaired trace-, but not delay-conditioned fear as evaluated by freezing behavior. A local cholinergic deafferentation of the EC using 192-IgG-saporin did not replicate this deficit, presumably because cholinergic interneurons were spared by the toxin. However, pretraining local blockade of EC muscarinic receptors with the M1 antagonist pirenzepine yielded a specific and dose-dependent deficit in trace-conditioned responses. The same microinjections performed after conditioning were without effect on trace fear responses. These effects of blocking M1 receptors are consistent with the notion that conditioned stimulus (CS)-elicited, acetylcholine-dependent persistent activities in the EC are needed to maintain a representation of a tone CS across the trace interval during the acquisition of trace conditioning. This function of the EC is consistent with recent views of this region as a short-term stimulus buffer.