Abstract mRNA transcripts for 3 isoforms of the α subunit of (Na,K)-ATPase have been peviously identified in the nervous system (designated α1, α2 and α3). In order to study the localization and expression of the different α isoforms in the peripheral nervous system, we prepared probes from the unique 3′ untranslated region of α1 cDNA, and from the translated region of α3 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, α1 mRNA was found in a discrete set of laterally placed motor neurons, while α3 was found in all the identified neurons of the spinal cord, including those motor neurons containing α1. In the lumbar DRG, α3 was uniformly distributed in DRG neurons, while α1 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 α1 probe in a perinuclear distribution, but contained no detectable α3. Dot blot analysis showed α1 and α3 in spinal cord and DRG, but only α1 in peripheral nerve. These results imply that: (1) the riboprobes are able to distinguish α1 from α3 isoform mRNAs under in situ hybridization conditions, (2) both α1 and α3 isoforms of (Na,K)-ATPase are found in neurons, (3) some motor and sensory neurons contain abundant α3 with little or no detectable α1, while others contain abundant α1 and α3, (4) no neurons in the lumbar spinal cord or DRG contain detectable α1 in the absence of α3, and (5) Schwann cells contain the α1 isoform but no detectable α3 isoform mRNA.