We have studied two gamma/delta T cell clones, E102 and E117, generated in a mixed lymphocyte culture using an allogeneic Epstein-Barr virus-transformed B cell line, E418. These clones were both found to express a molecular form of T cell receptor (TCR) infrequent in human peripheral blood, associating a V1-J1-C delta chain and a V3-JP2-C2 gamma chain. Functionally, they appeared as cytotoxic T lymphocytes (CTL) with non-major histocompatibility complex (MHC) (class I and II) requiring cytotoxicity, able to kill both the immunizing (i.e., E418) and unrelated (e.g., K562, REX, F601, and KAS) target cells. A monoclonal antibody, anti-10H3, able to selectively inhibit the cytotoxic activity of the clones has been produced. This reagent defines a 43-kD molecule, designated TCT.1, with broad distribution in the hematopoietic system, that appears to be distinct from class I MHC gene products. A series of functional experiments using various effector/target cell combinations strongly suggested that TCT.1 may represent a unique TCR ligand involved in the interaction between these particular CTL clones and certain of the target cells tested, while others were likely to be recognized and killed through a TCR-independent natural killer-like pathway. Although further experimentation will be needed to strengthen our interpretation of the present data, this study provides additional evidence that some T lymphocytes, in particular of the gamma/delta type, may interact specifically with target cells in a non-MHC class I/II-requiring fashion.