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Abstract: It has been proposed that the medial prefrontal cortex (mPFC) is not necessary for delay eyeblink conditioning (DEC). Here, we investigated the involvement of the mPFC in DEC with a soft or loud tone as the conditioned stimulus (CS) by using electrolytic lesions or muscimol inactivation of guinea pig mPFC. Interestingly, when a soft tone was used as a CS, electrolytic lesions of the mPFC significantly retarded acquisition of the conditioned response (CR), and muscimol infusions into mPFC distinctly inhibited the acquisition and expression of CR, but had no significant effect on consolidation of well-learned CR. In contrast, both electrolytic lesions and muscimol inactivation of mPFC produced no significant deficits in the CR when a loud tone was used as the CS, or in the unconditioned response (UR) when a soft or loud tone was used as the CS. These results demonstrate that the mPFC is essential for the DEC with the soft tone CS but not for the DEC with the loud tone CS. [Copyright &y& Elsevier]