Na,K-ATPase activity, Na,K-ATPase alpha- and beta-subunit mRNA abundance (mRNA alpha and mRNA beta), and gene transcription rates were determined in kidney cortex and liver of hypothyroid and triiodothyronine (T3)-treated rats. In hypothyroid rats, Na,K-ATPase activity (expressed per unit of DNA) was 3.6-fold greater in kidney cortex than liver, and the abundance of mRNA alpha and mRNA beta in kidney cortex exceeded that of liver by 2.8- and 5.2-fold, respectively. In vitro nuclear run-on analysis revealed similar rates of Na,K-ATPase alpha and beta gene transcription in nuclei isolated from either kidney cortex or liver. Administration of T3 for 72 h elicited a 2.3-fold stimulation of renal Na,K-ATPase activity that was associated with a 3.1- and 2.6-fold increase of mRNA alpha and mRNA beta content, respectively. In contrast, T3 induced a 1.3-fold stimulation of liver Na,K-ATPase activity accompanied by a 7.3-fold increase in mRNA alpha and no change in mRNA beta abundance. Transcription rates of alpha and beta genes (assayed by nuclear run-on) in renal cortex were both stimulated 1.8-fold in response to T3 injection. Similarly in liver nuclei, T3 treatment produced a 1.4- and 1.3-fold stimulation in the rate of alpha and beta gene transcription, respectively. These results indicate that significant discrepancies exist in the quantitative relationships between control and T3-induced changes in renal and hepatic enzyme activity, mRNA abundance and rate of gene transcription, and imply that the T3-induced increase in Na,K-ATPase abundance is mediated at both transcriptional and post-transcriptional steps.