Mononuclear cells (MNC) from normal humans consistently failed to give nonspecific cytotoxic responses. However, after removal of T cells by sheep erythrocyte (E) rosetting, the remaining non-RFC (rosette-forming cells) now gave significant nonspecific cytotoxic responses against both autologous and allogeneic target cells. Reconstitution experiments with T cell subpopulations failed to suppress these nonspecific non-E-RFC-mediated cytotoxic responses. There was also no evidence to indicate the involvement of antibody in this nonspecific cytotoxicity. The cytotoxic cells were characterized as non-E-rosetting, non-phagocytic, and glass adherent lymphocytes; no evidence of monocyte-macrophage participation was found. The inductive trigger of non-E-RFC-mediated cytotoxicity was found to be soluble factors released by T cells during E-rosette formation at 4 degrees C. Incubation of MNC with horse, marmoset and human erythrocytes under identical conditions failed to trigger cytotoxicity. The incubation of quiescent MNC with E-rosetting supernatants (ERS) induced nonspecific cytotoxic responses equivalent to those mediated by separated non-E-RFC. ERS-activated MNC destroyed both autologous and allogeneic target cells. The ERS supernatants themselves were not cytolytic. These findings suggested that cell separation procedures, and possibly in vivo events, which activate T cells may also induce non-T cell-mediated nonspecific cytotoxicity.