Four monoclonal antibodies (mAb) termed NKTA255, NKTA72, 1F1 and 1B1 were selected on the basis of their ability to inhibit the cytolytic activity of natural killer (NK) cell clones against P815 target cells. These mAb selectively reacted with normal or tumor cells of hematopoietic origin and displayed a cellular distribution similar to that of CD45 or CD11a/CD18 antigens. Immunoprecipitation experiments showed that they reacted with molecules with an apparent molecular mass of 40 kDa under both reducing and nonreducing conditions ("p40" molecules), thus differing from CD45 or CD11a/CD18 antigens as well as from the "inhibitory" receptors for HLA class I molecules (i.e. p58, CD94 and NKB1 molecules). Double-immunofluorescence analysis of peripheral blood mononuclear cells allowed the identification of three distinct populations on the basis of the fluorescence intensity of cells stained with anti-p40 mAb. p40bright cells were homogeneously HLA-DR-positive, p40medium cells were HLA-DR-negative but co-expressed CD56 antigens, while p40dull cells were all CD3+. Anti-p40 mAb strongly inhibited the lysis of K562 target cells, mediated by fresh NK cells, as well as the lysis of P815 target cells by NK or T cell clones. In addition, in redirected killing assays, anti-p40 mAb strongly reduced the anti-CD16 mAb-induced cytolytic activity of NK cell clones. On the contrary, they did not inhibit either the anti-CD3 or anti-T cell receptor mAb-mediated cytolytic activity of T cell clones or the lysis of allogeneic phytohemagglutinin blasts mediated by specific cytolytic T cell clones. The p40-induced inhibition of the NK cytotoxicity required optimal cross-linking, as anti-p40 mAb could inhibit the lysis of Fc gamma receptor (Fc gamma R)-positive but not of Fc gamma R-negative target cells. In addition, (Fab')2 fragments of anti-p40 mAb failed to inhibit the lysis of Fc gamma R-positive target cells. In conclusion, p40 molecules represent a new type of inhibitory surface molecule that appears to play a general regulatory role in the NK-mediated cytolysis.