Autoradiography with [14C]2-deoxyglucose was used to examine the functional activity of the rat brain during long- and short-term habituation of the acoustic startle reflex. Long-term habituated rats, when compared to short-term rats, showed an enhanced metabolic activation of the auditory system, with the exclusion of thalamocortical levels. Regional metabolic increases were also located within the cerebellum and its major input-output structures. In contrast, the midbrain reticular formation and its ascending thalamocortical activating system showed a widespread metabolic suppression. The findings provide the first map of structures in a mammalian brain with learning-related metabolic alterations dependent on long-term habituation.