A monoclonal antibody produced by a hybridoma cell line that has previously been shown to recognize an antigen present on murine macrophages and granulocytes (Mac-1) has now been shown to bind to human monocytes and polymorphonuclear leukocytes. Human monocytes bind about 40,000 M1/70 (anti-Mac-1) F(ab')2 or IgG molecules per cell in saturating conditions. In addition, M1/70 antibody recognizes a small population (less than 10%) of human blood lymphocytes. These cells express approximately 3-fold fewer Mac-1 antigen determinants than monocytes. Separation of this lymphocyte subset on a fluorescence-activated cell sorter has shown that all the natural killing activity in human blood can be found among these cells. Similarly, separation of the natural killer cells by an independent method based on their surface Fc receptor has shown that nearly all of them can be labeled by the hybridoma antibody. The same results are obtained when an F(ab')2 fragment of the M1/70 hybridoma antibody is used. The anti-Mac-1 antibody does not interfere with binding to the Fc receptor, nor does it interfere with either natural killing or antibody-dependent cellular cytotoxicity mediated by these cells. We conclude that there is a similar antigenic structure on the surface of murine and human monocytes and granulocytes and that this structure is also found on human natural killer cells.