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‐α (IFN‐α) 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‐α 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.