The sex differences in the functional contribution of brain substrates were explored following acquisition of a spatial working memory task using quantification of c-Fos protein. Rats of both sexes were trained during adolescence and adulthood in Morris water maze using a hidden escape platform with different daily location. Two control groups for each sex and age were added to explore the c-Fos activation not specific to the memory process. These were a free-swimming group (yoked control) and a handled control (CO) group. Behaviorally, no age differences were found in number of days required by males to acquire the task, but females showed a delay in acquisition during adolescence (P30) that improved in adulthood (P90). Both sexes showed a learning-related increase in Fos immunoreactivity in the anterodorsal and anteroventral thalamus and medial and lateral mammillary nuclei during adolescence. Higher levels of learning-related Fos immunoreactivity were found in the infralimbic cortex, CA3 and CA1 only in females. During adulthood the common activated region was the prelimbic cortex with the addition of the infralimbic cortex in the male group and the lateral mammillary nucleus in the female group. These results indicated sex and age differences in brain functioning following working memory task. However, they could not be necessarily linked with differences in performance since similar results were found between males and females during adulthood. The activation of common and interrelated structures suggests that these structures are involved in spatial processing but it also highlights the relevance of developmental changes for understanding the memory process.