The localization of sites of memory formation within the mammalian brain has proven to be a formidable task even for simple forms of learning and memory.Recent studies have demonstrated that reversibly inactivating a localized region of cerebellum, including the dorsal anterior interpositus nucleus, completely prevents acquisition of the conditioned eye-blink response with no effect upon subsequent learning without inactivation. This result indicates that the memory trace for this type of learning is located either (i) within this inactivated region of cerebellum or (ii) within some structure(s) efferent from the cerebellum to which output from the interpositus nucleus ultimately projects. To distinguish between these possibilities, two groups of rabbits were conditioned (by using two conditioning stimuli) while the output fibers of the interpositus (the superior cerebellar peduncle) were reversibly blocked with microinjections of the sodium channel blocker tetrodotoxin. Rabbits performed no conditioned responses during this inactivation training. However, training after inactivation revealed that the rabbits (trained with either conditioned stimulus) had fully learned the response during the previous inactivation training. Cerebellar output, therefore, does not appear to be essential for acquisition of the learned response. This result, coupled with the fact that inactivation of the appropriate region of cerebellum completely prevents learning, provides compelling evidence supporting the hypothesis that the essential memory trace for the classically conditioned eye-blink response is localized within the cerebellum.Identification of the site or sites of memory formation and storage within the brain for any particular type of memory is an essential prerequisite for elucidating the cellular or subcellular mechanisms as well as the network level properties that mediate the acquisition, retrieval, and expression of that particular memory. Although much progress toward identifying potential sites of memory formation and storage has been achieved, definitive localization of a particular memory locus within the mammalian brain has remained frustratingly elusive. A major obstacle impeding this localization of memory traces is the necessity of first identifying the neural-anatomical circuitry essential for acquisition and expression of a particular learned response. For at least one form of learned behavioraversive, classically conditioned discrete skeletal movements, specifically, the classically conditioned eye-blink responsethe neural circuitry essential for acquisition and expression of the learned response has largely been identified (for review, see ref. 1). In brief, the results of lesion, recording, and stimulation studies indicate that the conditioned stimulus (CS) pathway includes sensory relay nuclei, the pontine nuclei, and mossy fiber projections, via the middle cerebellar peduncle, toThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be he...