Calmodulin (CaM), the ubiquitous intracellular calcium-binding protein, is coded by three bona fide CaM genes (CaM I, CaM II and CaM III) in mammals. They code for the same protein and are transcribed at particularly high levels in the brain, where CaM plays an essential role in basic neuronal functions. In this study, the expression of the three CaM genes in response to osmotic stimuli by water deprivation was investigated in the rat brain, with particular interest as concerns the hypothalamic magnocellular nuclei. CaM mRNA levels were determined by quantitative in situ hybridization autoradiography with gene-specific [35S]cRNA probes. In response to osmotic challenge, it was found that upregulation of the three CaM genes participates in the activation of the hypothalamo-hypophyseal system in the supraoptic nucleus (SON) (126% to 169%), but not in the magnocellular part of the paraventricular hypothalamic nucleus (PVN) (-10%). CaM mRNA levels decreased by 10%-15% in the suprachiasmatic nucleus (SCh) and many other extrahypothalamic brain areas. The opposite responses of the CaM gene expression in the SON and the magnocellular part of the PVN suggest a functional difference between them. Moreover, the significantly different magnitudes of the changes in the CaM mRNA levels in the SON nucleus (138%, 126% and 169% for CaM I, CaM II and CaM III, respectively) exemplify the precise differential control of the CaM gene expression in the brain.