The Na+,K'-ATPase plays a key role in the regulation of ion fluxes and membrane repolarization in the CNS . We have studied glucocorticoid effects on biosynthesis of the Na+,K+-ATPase and on ouabain binding in the ventral horn of the spinal cord using intact rats, adrenalectomized (ADX) rats, and ADX rats receiving dexamethasone (ADX + DEX) during 4 days . Cryostat sections from spinal cords were incubated with a 35Soligonucleotide coding for the a3-subunit or a 3H-cDNA coding for the 01-subunit of the Na+,K+-ATPase using in situ hybridization techniques . In ventral horn motoneurons, grain density per cell and grain density per area of soma for both probes were slightly reduced in ADX rats but significantly increased in the ADX + DEX group, using ANOVA and the Bonferroni's test . Statistical analysis of frequency histograms of neuronal densities further indicated a significant shift to the right for intact rats compared with ADX rats for both probes . Concomitantly, [3 H]ouabain binding to membrane preparations from ventral horns was reduced in ADX rats and restored to normal by DEX administration . No effect of adrenalectomy or DEX treatment was obtained in the dorsal horn . In conclusion, glucocorticoids positively modulate the mRNA for the a3-subunit and the Q1 -subunit of the Na+,K+-ATPase and recover ouabain binding to normal values . The increments of the synthesis and activity of an enzyme affecting membrane repolarization and synaptic neurotransmission are consistent with the alleged stimulatory effect of glucocorticoids on spinal cord function .