Cellular calcium signaling plays important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. In epithelial cells calcium signaling is initiated mainly by calcium release from endoplasmic reticulum-associated intracellular calcium pools. Because calcium is accumulated in the endoplasmic reticulum by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA), these enzymes play a critical role in the control of calcium-dependent cell activation, growth and survival. We investigated the modulation of SERCA expression and function in human lung adenocarcinoma cells. In addition to the ubiquitous SERCA2 enzyme, the SERCA3 isoform was also expressed at variable levels. SERCA3 expression was selectively enhanced during cell differentiation in lung cancer cells, and marked SERCA3 expression was found in fully differentiated normal bronchial epithelium. As studied by using a recombinant fluorescent calcium probe, induction of the expression of SERCA3, a lower calcium affinity pump, was associated with decreased intracellular calcium storage, whereas the amplitude of capacitative calcium influx remained unchanged. Our observations indicate that the calcium homeostasis of the endoplasmic reticulum in lung adenocarcinoma cells presents a functional defect due to decreased SERCA3 expression that is corrected during pharmacologically induced differentiation. The data presented in this work show, for the first time, that endoplasmic reticulum calcium storage is anomalous in lung cancer cells, and suggest that SERCA3 may serve as a useful new phenotypic marker for the study of lung epithelial differentiation.