We previously reported that thyroid hormone, 3,3′,5-triiodo-L- thyronine (T3), increased Na,K-ATPase activity of adult rat alveolar epithelial cells in a transcription-independent manner via increased cell surface expression of the α1 and β1 subunits of Na,K-ATPase. Now we sought to identify signaling molecules necessary for T3 stimulation of Na,K-ATPase activity in alveolar epithelial cells. Whereas protein kinase A inhibitor H-8 and protein kinase C inhibitor bisindolymaleimide did not block the T3-induced increase in Na,K-ATPase activity, two inhibitors of phosphoinositide 3-kinase (PI3K), wortmannin and Ly294002, and two Src kinase inhibitors, PP1 and PP2, blocked the T3-induced Na,K-ATPase activity. T3 stimulated the activity of PI3K as measured by phosphatidylinositol 3-phosphate. T3 also stimulated the serine 473 phosphorylation of the PI3K downstream molecule PKB/Akt in a dose-dependent manner. Transient expression of a constitutively active mutant of the PI3K catalytic subunit p110 augmented Na,K-ATPase activity and increased the amount of cell surface Na,K-ATPase α1 subunit protein. T3 also stimulated Src family kinase activity. Transient expression of a constitutively active Src kinase increased Na,K-ATPase activity, PI3K activity, and phosphorylation of PKB/Akt at serine 473. PP1 or PP2 blocked T3-stimulated PKB/Akt phosphorylation at serine 473 and PI3K activity that was activated by an active mutant of Src; however, wortmannin did not inhibit the T3-stimulated Src kinase activity. Although PP1 and wortmannin abolished the increase in Na,K-ATPase activity induced by the active mutant of Src, PP1 did not inhibit the active mutant of PI3K-up-regulated Na,K-ATPase activity. In summary, T3 stimulates the PI3K/PKB pathway via the Src family of tyrosine kinases, and activation of both the Src family kinases and PI3K is required for the T3-induced stimulation of Na,K-ATPase activity and its cell surface expression in adult rat alveolar epithelial cells.