Human peripheral blood natural killer (NK) cells (CD56+, CD16+, CD3 epsilon- lymphocytes) express CD69 after their stimulation by interleukin-2 (IL-2) or interferon-alpha (IFN-alpha). This activation antigen represents a triggering surface molecule in NK cell clones as its stimulation triggers the cytolytic machinery of these cells. However, the mechanisms regulating the expression of CD69 in NK cells are unknown despite the functional relevance of CD69 in NK cell activation. Thus, we have analyzed the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the expression of CD69 on purified NK cells activated by IL-2, IFN-alpha, anti Fc gamma RIII (CD16) monoclonal antibodies or by K562 target cells. We found that CD69 is induced on NK cells not only by IL-2 and IFN-alpha but also by activation of the CD16 pathway, the interaction with NK target cells and the direct activation of PKC by phorbol 12-myristate 13-acetate (PMA), indicating that CD69 induction is associated to different NK activation pathways. The treatment with the PKC inhibitor staurosporine abolished the induction of CD69 induced by PMA or K562. However, it did not significantly affect CD69 induction by IL-2, IFN-alpha or CD16 cross-linking. This demonstrates that whereas PKC can play a central role in the regulation of CD69 expression in some instances (response to K562 cells or PMA), it does not participate in others (response to IL-2, IFN-alpha or anti CD16 monoclonal antibodies). On the other hand genistein, a competitive inhibitor of PTK enzymes, blocked the expression of CD69 induced by activation of NK cells via IL-2 or IFN-alpha receptors, CD16 and K562 receptor(s), indicating that stimulation of PTK is a common step in the signal transduction events leading to the induction of CD69 antigens after the activation of NK cells via these receptors.