The purpose of the study was to map the functional neuroanatomy of simple associative learning in humans. Eyeblink conditioning was studied in eight normal volunteers using positron emission tomography and H2150.Regional cerebral blood flow was assessed during three sequential phases: (i) explicitly unpaired presentations of the unconditioned stimulus (air puff to the right eye) and conditioned stimulus (binaural tone), (ii) paired presentations of the two stimuli (associative learning), and (Wi) presentation of the conditioned stimulus alone. During associative learning, relative to the unpaired phase, blood flow was significantly increased in primary auditory and left posterior cingulate cortices and significantly decreased in areas of the right cerebellar, right prefrontal, right parietal, and insular cortices and right neostriatum. The lateralization of the changes may relate to the functional organization of memory and learning processes in the brain. The activation in primary auditory cortex is an example, using a neuroimaging technique, of a learning-related change in primary sensory cortex in humans. The changes in areas such as the cerebellum, prefrontal cortex, and neostriatum provide support for their roles in associative learning as proposed by animal models. Moreover, these findings show that in humans, even simple classical conditioning involves distributed changes in multiple neural systems.The behavioral laws underlying classical conditioning of skeletal responses, such as eyeblink, are well defined, making this paradigm especially attractive for studying human associative learning (1, 2). Although animal studies of associative learning have provided considerable information on neurophysiological and neuroanatomical substrates (3-7), little is known about the neural systems involved in associative learning in humans.