A monoclonal IgM antibody (HNK-1) was produced against a membrane antigen from the cultured T cell line, HSB-2. By indirect immunofluorescence, this antibody reacted with certain human cultured T cell lines (HSB-2 and MOLT-4 but not MOLT-3) but not with other lines of B cell or phagocytic cell origin. HNK-1 reacted with 15.1 +/- 7.1% of normal blood lymphocytes but was unreactive with monocytes, granulocytes, erythrocytes, and platelets. HNK-1+ cells separated by a fluorescence-activated cell sorter (FACS) were a homogeneous population of medium sized lymphocytes with abundant neutrophilic cytoplasm containing azurophilic granules. HNK-1+ cells were nonadherent, surface Ig-, mostly FcIgG receptor+ and both positive and negative for demonstrable sheep erythrocyte (E) rosetting capability. Cell suspensions enriched for E-rosetting T cells and depleted of FcIgG receptor+ cells contained few (6%) HNK-1+ cells. Depletion of HNK-1+ cells from blood mononuclear cell populations by complement (C) mediated lysis greatly reduced NK activity against K-562 target cells and K cell lytic activity against antibody-coated chicken red blood cells. Treatment with HNK-1 alone or C alone did not affect these activities. When the FACS was utilized to separate HNK-1+ and HNK-1- cells from 6 individuals, the HNK-1+ cell population contained almost all of the NK and K cell function. The monoclonal antibody HNK-1 thus defines the first differentiation antigen shown to be selectively expressed on human NK and K cells.