In asthmatic patients, eosinophils massively infiltrate the lung tissues and migrate through lung epithelium into the airways. The regulatory mechanisms involved are obscure. We studied the role of calcium in the migration of human eosinophils across monolayers of human lung epithelial H292 cell line cells induced by combined chemotactic solutions of platelet-activating factor and C5a. The transepithelial migration of eosinophils was attenuated by depletion of the external Ca2+ in the migration system, whereas the eosinophil migration itself was unaffected as evidenced by measuring eosinophil chemotaxis in the Boyden chamber in the absence of epithelial cells. Buffering of intracellular Ca2+ in eosinophils with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) inhibited both eosinophil transepithelial migration and eosinophil chemotaxis in the Boyden chamber, suggesting the importance of intracellular Ca2+ in eosinophil transmigration. Although loading of BAPTA/AM or addition of thapsigargin to the epithelial cells effectively changed their cytoplasmic free Ca2+ concentrations, neither of these treatments affected transepithelial migration of eosinophils. Interestingly, addition of La3+ (0.2 mM) to epithelial cells suppressed eosinophil transmigration whereas addition of La3+ to eosinophils did not. Taken together, these results show the importance of Ca2+ in eosinophil migration across lung epithelium and support a distinctive regulatory role of intracellular and extracellular Ca2+ for the two cell types involved in this process; i.e., the transmigration of human eosinophils across a monolayer of lung epithelial cells is regulated by the intracellular Ca2+ in eosinophils, whereas the ability of the lung epithelial cell monolayer to allow eosinophil passage is dependent on the extracellular Ca2+.