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1. mRNA transcripts for three isoforms of the alpha subunit of (Na,K)-ATPase have been previously identified in the rat nervous system and designated alpha 1, alpha 2 and alpha 3. 2. In order to study the localization and expression of the different alpha isoform mRNAs on a regional and cellular level in the brain, we prepared probes from the unique 3' untranslated region of rat alpha 1 cDNA and from a segment containing a portion of the translated region of rat alpha 3 cDNA. These probes were used in dot blot and in situ hybridization assays of rat brain. 3. alpha 1 mRNA was found predominantly in cerebral cortex, dentate gyrus of hippocampus, and specific isolated brain-stem nuclei such as locus ceruleus and motor nuclei V and VII. In contrast alpha 3 mRNA was found predominantly in pyramidal neurons in the deep layers of cerebral cortex, in both pyramidal and dentate gyrus neurons of the hippocampus, and in neurons of most subcortical structures of the thalamus, basal ganglia, and brain-stem nuclei. 4. In the cerebellum, Purkinje cells showed predominantly alpha 3, as did stellate and basket cells. The granule cells contained predominantly alpha 1. 5. These experiments show that mRNAs for both alpha 1 and alpha 3 isoforms of (Na,K)-ATPase are found in neurons of the CNS. The isoforms have unique cellular and regional distributions, which in some cases overlap.