Cytosolic free Ca2+ plays important roles in the regulation of physiological processes in tracheal epithelial cells and is probably regulated by many ion-transporting ATPases in these cells. Therefore, the effect of vanadate was investigated to characterize microsomal ion-transporting ATPases. Dose response experiments showed that vanadate had a biphasic effect on the microsomal ATPase activity: a decrease at the vanadate concentration below 100 microM, and a steep decrease at the concentration above 100 microM. The dose response data were fitted to two sigmoidal functions, corresponding to a low-affinity vanadate-sensitive (LAVS) ATPase and a high-affinity vanadate-sensitive (HAVS) ATPase. In 45Ca2+ uptake experiments, both LAVS and HAVS ATPases mediated microsomal 45Ca2+ uptake. The LAVS ATPase was selectively sensitive to thapsigargin in both ATPase activity and 45Ca2+ uptake, suggesting that it is an ER/SR-type intracellular Ca2+-ATPase. Although the HAVS ATPase mediated one-fourth of microsomal 45Ca2+ uptake, its activity was not sensitive to thapsigargin. These results indicate that the activities of these two vanadate-sensitive ATPases are mediated by different enzymes, since thapsigargin only blocks the activity of LAVS ATPase. In conclusion, there are two types of vanadate-sensitive microsomal ATPases, and these ATPases mediate microsomal 45Ca2+ uptake in airway epithelial cells.