The in vitro demonstration of the ability of NK cells to secrete cytokines prompted in vivo studies that illustrated the importance of NK cell-derived cytokines in regulating immune responses. Cross-linking of CD16 on NK cells can stimulate cytokine production. CD16-independent interactions capable of stimulating cytokine production have also been described, but molecules mediating such stimulations remain to be biochemically defined. We report here that cross-linking of CD45 specifically stimulates IFN-gamma production in human NK cells. The NK cells used were IL-2-activated adherent NK cells and from the NK3.3 cell line. The ability of CD45 mAbs to stimulate NK cells appears not to be dependent on CD16, as CD45 mAbs of both IgG1 and IgG2a isotypes were equally stimulatory, as were F(ab')2 compared with whole anti-CD45 mAbs. Resting NK cells, like T cells, express predominantly CD45RA, whereas IL-2 activated adherent NK cells acquire expression of CD45RO. Abs specific for CD45RO, but not CD45RA, were able to stimulate IFN-gamma production in NK cells. It has been reported that one ligand for CD45RO is CD22 beta. We tested the ability of CD22-expressing transfectants to bind to and stimulate NK cells. Whereas NK cells bound to CD22 alpha and CD22 beta transfectants, this interaction was not inhibited by CD45RO Abs. In addition, neither of the CD22-transfectants were able to stimulate NK3.3 cells to secrete IFN-gamma. These observations collectively suggest that binding of NK3.3 cells to CD22 may be independent of CD45RO on NK3.3 cells.