Calmodulin is a major calcium-binding protein in the mammalian brain, playing an important role in neuronal cell function. Its amino acid sequence is highly conserved and the protein is encoded by multiple genes. In the mouse brain, as well as in the rat and the human brain, three different genes have been detected for calmodulin, CaM I, CaM II and CaM III, all of which encode an identical protein. We studied the pattern of expression of the three calmodulin genes and the pattern of calmodulin distribution in the mouse brain by in situ hybridization histochemistry and immunohistochemistry. We found that calmodulin messenger RNAs from the three calmodulin genes were widely expressed in the mouse brain. Nevertheless, there were differences in their patterns of distribution. In general, all calmodulin messenger RNAs were preferentially distributed in hippocampus, cerebral cortex and cerebellar cortex, and CaM II messenger RNA also in caudate-putamen. However, all messenger RNAs showed clearly differentiated patterns of distribution in the hippocampus and the cerebellar cortex. Calmodulin immunoreactivity was present in all cells so far examined. Immunostaining was observed both in the cell nucleus, where it was especially strong, and in the cytoplasm. Our results suggest that the three calmodulin genes are differentially regulated in the mouse brain and also that, although all calmodulin genes have a basal expression, precise regulation of calmodulin levels might be attained through the different contribution of the three calmodulin genes.