mRNA transcripts for 3 isoforms of the alpha subunit of (Na,K)-ATPase have been previously identified in the nervous system (designated alpha-1, alpha-2 and alpha-3). In order to study the localization and expression of the different alpha isoforms in the peripheral nervous system, we prepared probes from the unique 3' untranslated region of alpha-1 cDNA, and from the translated region of alpha-3 cDNA. These probes were used in dot blot and in situ hybridization assays of rat spinal cord, dorsal root ganglia (DRG), and sciatic nerve. Within the ventral horn of lumbar spinal cord, alpha-1 mRNA was found in a discrete set of laterally placed motor neurons, while alpha-3 was found in all the identified neurons of the spinal cord, including those motor neurons containing alpha-1. In the lumbar DRG, alpha-3 was uniformly distributed in DRG neurons, while alpha-1 was abundant in some neurons but little or none was found in other neurons. Satellite cells contained neither isoform. Schwann cells in sciatic nerve were labeled with the alpha-1 probe in a perinuclear distribution, but contained no detectable alpha-3. Dot blot analysis showed alpha-1 and alpha-3 in spinal cord and DRG, but only alpha-1 in peripheral nerve. These results imply that: (1) the riboprobes are able to distinguish alpha-1 from alpha-3 isoform mRNAs under in situ hybridization conditions, (2) both alpha-1 and alpha-3 isoforms of (Na,K)-ATPase are found in neurons, (3) some motor and sensory neurons contain abundant alpha-3 with little or no detectable alpha-1, while contain abundant alpha-1 and alpha-3, (4) no neurons in the lumbar spinal cord or DRG contain detectable alpha-1 in the absence of alpha-3, and (5) Schwann cells contain the alpha-1 isoform but no detectable alpha-3 isoform mRNA.