We studied the mechanisms underlying the natural tolerance of thymus-derived (T) lymphocytes for self-antigens. Lymphocytes from the thymus or lymph nodes of inbred rats were autosensitized in vitro against monolayers of autochthonous thymus reticulum cells or syngeneic fibroblasts. Receptors for self-antigens were detected by the specific adherence of normal lymphocytes to syngeneic cells. The achievement of active cell-mediated autosensitization was assayed by measuring the immunospecific lysis of syngeneic target cells in vitro, or graft-versus-host (GvH) reactions in vivo. The following observations were made using these systems. (a) A fraction of normal lymphocytes was found to have specific surface receptors that are able to recognize self-antigens which seem to be accessible in vivo. These potentially self-reactive lymphocytes were activated by incubation with syngeneic or autochthonous cells in vitro. Hence, the elimination of potentially self-reactive lymphocytes cannot be the only basis for natural self-tolerance. Therefore, the maintenance of self-tolerance in vivo appears to involve suppression of the immune reactivity of such self-tolerant lymphocytes. (b) We found that control of autosensitization depends upon the inhibition of the recognition of self-antigens. A GvH reaction in vivo could not be suppressed once recognition of self-antigens had occurred in vitro. Moreover, studies of the kinetics of antigen recognition indicated that several hours of incubation in vitro were needed for the inactivation of factors specifically inhibiting self-recognition. (c) We found that factors which inhibit self-recognition are present in fresh autologous serum. Treatment of the lymphocytes, but not syngeneic adsorbing cells, with autologous serum prevented recognition of syngeneic antigens. Allogeneic serum did not prevent self-recognition, and autologous serum did not inhibit the recognition of foreign antigens. These findings indicate that natural tolerance of T lymphocytes to self-antigens can be regulated by serum factors which act on the lymphocytes. The immunospecificity of the inhibitory effect suggests that these factors may be soluble self-antigens in a tolerogenic form.