The biochemical and immunological properties of a cell toxin(s) released into the fluid phase and expressed on the surface of lectin-activated alloimmune human cytotoxic lymphocytes has been investigated. The results indicate that the initial cytotoxin(s) released into the supernatant from alloimmune lymphocytes represents a precursor form of lymphotoxin (pre-LT), and in this precursor form, the α and β antigens exist as masked or cryptic determinants. The pre-LT form in unfractionated supernatants was defined immunologically by neutralization with a polyspecific anti-LT antisera (anti-WS), and its lack of reactivity with anti-α and anti-β LT antisera. However, gel filtration chromatographic profile of the pre-LT cytotoxic activity was heterogeneous and showed multiple m.w. classes that were characteristic of the chromatograms of traditionally defined LT. These fractionated components were now readily neutralized with anti-α LT antiserum. In addition, the pre-LT cytotoxic activity in unfractionated supernatants treated by various physical or chemical means rendered the cytotoxic activity neutralizable by an anti-α LT antiserum, indicating an immunologic relationship between the pre-LT form and components of the LT system. The antigens associated with pre-LT form were detectable, in part, on the LT-Cx and γ LT molecules by a fluid phase immunoadsorption assay. A functional immunoadsorption assay was used to detect the antigenic determinants of the pre-LT form on the cell surface of alloimmune lymphocytes before and after lectin activation. α LT-associated antigens were detectable on cytotoxic effectors only after lectin activation. These results imply LT may function as cell surface delivered cytotoxic molecules. LT activity was not detected in supernatants obtained from alloimmune cytotoxic reactions, however, the pre-LT and α-LT neutralizing activities of the anti-LT sera were significantly diminished after the incubation of these sera in the cytotoxic reaction and indicated that LT molecules were produced during the cytotoxic reaction, and LT remained closely associated with the lymphocyte:target cell conjugates. The relationship of the pre-LT form to the capacity of the various anti-LT antisera to affect the human alloimmune cytotoxic reaction is discussed. The results provide additional support to the concept that the LT system is involved in the lytic mechanism of cytotoxic T cells.