The avian eggshell gland (ESG) is a tissue specialized in transporting the Ca²⁺ required for eggshell formation and represents a unique biological system in which the calcification process takes place in a circadian fashion. With the use of RNA fingerprinting, a set of genes differentially induced at the time of calcification was detected, one of which was identified as the ₁-subunit of Na⁺-K⁺-ATPase. The gene was expressed in a circadian manner in both cell types populating the ESG, but in different temporal patterns, suggesting distinct mechanisms of regulation. Ca²⁺ flux and mechanical strain were found to regulate gene expression in the inner glandular epithelium and the pseudostratified epithelium facing the lumen, respectively. Mechanical strain also affected gene expression in cell layers facing the lumen in other parts of the oviduct. Only the ₁-isoform, not the ₂- or ₃-isoform, of Na⁺-K⁺-ATPase was expressed in the ESG. In summary, we demonstrate that the ₁-subunit Na⁺-K⁺-ATPase gene is expressed in different epithelial cell types in the ESG and is regulated by various mechanisms, which may reflect the disparity in the physiological roles of the cells in the process of eggshell formation.