CD53 is a member of a novel family of molecules with four presumably membrane-spanning domains. The structure and functional characteristics of these molecules indicate that they may play an important role in transmembrane communication. We therefore investigated whether CD53 is involved in activation of human leukocytes. Cross-linking of cell-bound F(ab')2 fragments of two different anti-CD53 mAb with F(ab')2 anti-mouse Ig led to cytoplasmic calcium fluxes in B cells, monocytes, and granulocytes and activation of the monocyte oxidative burst. These responses were specific for CD53, as cross-linking of CD11a, CD18, CD35, CD43, CD44, CD45, or CDw50 did not induce leukocyte activation. Low concentrations of staurosporine (10 to 20 nM) completely inhibited PMA-mediated activation, but had no effect on CD53-mediated calcium fluxes and inhibited only partially CD53-mediated oxidative burst. This suggests that CD53-mediated signaling is largely independent of protein kinase C. CD53-mediated calcium fluxes were inhibited by high concentrations of staurosporine (300 to 500 nM) but not by ADP-ribosylating toxins, suggesting dependence on tyrosine kinases rather than GTP-binding proteins. The results indicate that CD53, like several other leukocyte Ag with four membrane-spanning regions, has the ability to mediate cell activation, and support the view that these molecules are involved in transmembrane communication.