CD69 is a differentiation antigen expressed shortly after activation on T lymphocytes and other cells of haematopoietic origin, including natural killer (NK) cells. The function of CD69 on T lymphocytes acting as a costimulatory molecule in proliferation and lymphokine secretion is well established. NK cells express CD69 after activation by different stimuli such as phorbol 12-myristate 13-acetate (PMA), interleukin (IL)-2, IL-12, interferon-alpha (IFN-alpha) or anti-CD16 monoclonal antibodies (mAbs). However, although it has been shown that CD69 triggers NK-cell-mediated cytolytic activity, its effect on other NK-cell functions has not been studied. Furthermore, the possible interaction of CD69 triggering with other C-lectin type inhibitory receptors is not known. Thus, the objective of this work is to determine whether CD69-mediated NK cytotoxicity can be regulated by CD94 inhibitory receptor and the role of CD69 on other NK-cell functions different of cytotoxicity. The results show that CD69-mediated NK cytotoxicity can be abrogated by CD94 stimulation in NK cells expressing the CD94 inhibitory form of the receptor, indicating that CD94 regulates the cytotoxic events initiated by a wide variety of NK activatory receptors. We also show that anti-CD69 mAbs, not only triggered NK cytotoxicity, but also induce NK-cell proliferation, CD25 and intracellular adhesion molecule-1 (ICAM-1) expression, TNF-alpha production and Ca2+ mobilization in preactivated NK cells. These results suggest that CD69 plays a crucial role in NK-cell function contributing to sustain NK-cell activation, as it has been previously demonstrated in T cells.