Human lymphocytes with natural killer (NK) activity, including most activated γ/δ+ T lymphocytes, recognize and lyse tumor target cells without requiring recognition of major histocompatibility complex antigen. However, unlike γ/δ+ T lymphocytes, NK cells do not express CD3/T cell receptor (TCR) molecules, and the receptors involved in cell-mediated cytotoxicity are unknown. To further delineate circulating NK cells, we developed monoclonal antibodies (mAbs) against the human NK leukemia YT2C2. We report the isolation of a mAb termed BY55, recognizing at the cell surface a novel 80-kD protein with restricted expression, In addition to the immunizing cell line, this mAb binds to circulating NK cells, γ/δ+ cells, and a minor subset of ot/B+ T lymphocytes. Expression of the BY55 mAb-reactive epitope/ molecule is regulated by activation, as short-term culture of peripheral blood lymphocytes (PBL) with phorbol ester induced its downmodulation. Furthermore, BY55 mAb reactivity was found neither with the NK nor with the TCR α/β+ and γ/δ+ clones tested. Biochemical studies as well as phenotypic analysis revealed that this structure is different from all previously identified molecules on the lymphocyte cell surface. Interestingly, we found that BY55+ cells exert most NK activity obtained with fresh circulating lymphocytes. We report that within fresh E rosette-positive PBL only a subset of the CD16+, CD56+, and CD57+ cells coexpressed BY55 molecule, indicating that BY55 mAb defines a unique subset mediating NK activity of circulating PBL.