The purpose of this study was to examine the effects of protein synthesis inhibition in the intermediate cerebellum on the acquisition and expression of classically conditioned nictitating membrane responses in the rabbit. Animals were conditioned for three days in a standard delay paradigm. Before each training session, either a solution of anisomycin (a protein synthesis inhibitor) or vehicle was bilaterally injected into the interposed cerebellar nuclear. Following these three training sessions, rabbits were tested to determine whether the previous training under the influence of anisomycin or vehicle resulted in the acquisition of conditioned responses. In this test, animals that were injected previously with the protein synthesis inhibitor exhibited significantly less retention of conditioned responses than rabbits injected with vehicle. Additional experiments demonstrated that anisomycin does not block the expression of conditioned responses during conditioning or in well-trained animals. Microinjections of muscimol at the same sites of the previous drug infusions suppressed the expression of conditioned responses, indicating that the protein synthesis inhibitor was applied to the eyeblink-related parts of cerebellar circuits. The obtained data are the first to demonstrate that a manipulation of cerebellar circuits, which does not affect the performance of learned behavior, can affect the process of learning. These results suggest that the synthesis of new proteins in the intermediate cerebellum participates in the formation of plastic changes responsible for eyeblink conditioning.