Late in gestation, the developing airspace epithelium switches from chloride and fluid secretion to sodium and fluid absorption. Absorption requires Na,K-ATPase in combination with apical sodium entry mechanisms. Hypothyroidism inhibits perinatal fluid resorption and T3 stimulates adult alveolar epithelial cell (AEC) Na,K-ATPase. This study explored the developmental regulation of Na,K-ATPase by T3 in fetal rat distal lung epithelial (FDLE) cells. T3 increased Na,K-ATPase activity in primary FDLE cells from gestational day E19 and the FD19 cell line. However, T3 did not increase the Na,K-ATPase activity in less mature FDLE cells, including primary E17 and E18 FDLE cells and the FD18 cell line. T3 signal pathways were compared between the late FDLE and adult AEC to determine the site of the switch in responsiveness to T3. As in adult AEC, in FD19 cell line the PI3K inhibitor wortmannin blocked the T3-induced increase in Na,K-ATPase activity and plasma membrane quantity. T3 caused a parallel increase in phosphorylation of Akt at Ser-473 in FDLE cells from E19, but not from E17 or E18. In the FD18 cell line, transient expression of a constitutively active PI3K catalytic p-110 subunit significantly augmented the Na,K-ATPase activity and the cell surface expression of Na,K-ATPase alpha1 protein. In conclusion, FDLE cells from E17 and E18 lacked T3 sensitive Na,K-ATPase activity, but acquired this response at E19. The developmental stimulation of Na,K-ATPase by T3 in rat FDLE cells requires activation of PI3K and the acquisition of T3 responsiveness may be at PI3K or upstream in the signaling pathway.