To investigate brain substrates of spatial memory, the cellular expression of c-Fos protein in rats was studied after training the animals to perform a spatial reference memory task and a working reference memory task in a Morris water maze. The number of c-Fos positive neuronal nuclei was quantified in several brain regions: entorhinal cortex, anterior cingulate gyrus, and supramammillary nucleus. The results showed that spatial training in reference and working memory tasks increased the number of entorhinal cortex activated neurons (c-Fos positive neurons). No clear association was found between c-fos activation in the anterior cingulate gyrus and either spatial reference or working memory tasks. The number of c-Fos immunoreactive neuronal nuclei in the supramammillary neurons was greater in the spatial working memory groups than in the spatial reference memory groups suggesting that neu - rons of the supramammillary nucleus plays an important role in spatial processing.