Relative cerebral glucose metabolism was examined with positron-emission tomography (PET) as a measure of neuronal activation during performance of the classically conditioned eyeblink response in 12 young adult subjects. Each subject received three sessions: (i) a control session with PET scan in which unpaired presentations of the tone conditioned stimulus and corneal airpuff unconditioned stimulus were administered, (ii) a paired training session to allow associative learning to occur, and (iii) a paired test session with PET scan. Brain regions exhibiting learningrelated activation were identified as those areas that showed significant differences in glucose metabolism between the unpaired control condition and well-trained state in the 9 subjects who met the learning criterion. Areas showing significant activation included bilateral sites in the inferior cerebellar cortex/deep nuclei, anterior cerebellar vermis, contralateral cerebellar cortex and pontine tegmentum, ipsilateral inferior thalamus/red nucleus, ipsilateral hippocampal formation, ipsilateral lateral temporal cortex, and bilateral ventral striatum. Among all subjects, including those who did not meet the learning criterion, metabolic changes in ipsilateral cerebellar nuclei, bilateral cerebellar cortex, anterior vermis, contralateral pontine tegmentum, ipsilateral hippocampal formation, and bilateral striatum correlated with degree of learning. The localization to cerebellum and its associated brainstem circuitry is consistent with neurobiological studies in the rabbit model of eyeblink classical conditioning and neuropsychological studies in braindamaged humans. In addition, these data support a role for the hippocampus in conditioning and suggest that the ventral striatum may also be involved.Eyeblink conditioning is a basic form of associative learning that has been studied extensively in humans since the 1930s. Animal models of this form of conditioning have demonstrated close behavioral parallels with human conditioning, lending support to the hypothesis that the learning shares common neurobiological substrates in humans and other mammals (1). Eyeblink conditioning is well-suited for neurobiological analyses because the use of appropriate unpaired control procedures can help distinguish between areas of the brain that are responding to the sensory stimuli or the motor response and those that are selectively activated under paired learning conditions. This feature makes the paradigm an ideal candidate for neurobiological analysis in humans using positron-emission tomography (PET) to identify regions of the brain that are specifically activated under paired training conditions relative to unpaired control procedures.Although eyeblink conditioning is a seemingly simple form of learning, under normal conditions it appears to involve the interaction of diverse brain systems. Neurobiological studies (conducted primarily in rabbits) have identified the cerebellum and its brainstem connections as essential components of the eyeblink condit...