Recent studies have demonstrated that noncytolytic T-cells can mediate regression of murine tumors. In this report, we demonstrate that MCA-105 tumor-draining lymph node cells (DLN) activated with the protein kinase C activator, bryostatin 1, plus a calcium ionophore are capable of inducing specific tumor regression in vivo when adoptively transferred to mice with established metastases. However, these activated DLN cells lack in vitro cytotoxicity against autologous tumor. Antibody against gamma-interferon (IFN-gamma) markedly inhibited the therapeutic efficacy of these activated DLN cells. Anti-tumor necrosis factor produced a statistically significant but weaker inhibition of tumor regression. IFN-gamma, but not tumor necrosis factor alpha, could be shown to be secreted by activated DLN cells in vitro in response to specific tumor. Secretion of IFN-gamma was primarily a function of CD8+ T-cells. IFN-gamma was not directly cytotoxic to sarcoma cells in vitro. Moreover, tumor cells incubated with IFN-gamma were not more susceptible to lysis by activated DLN cells. However, recombinant murine IFN-gamma had a significant antiproliferative effect against MCA-105 tumor cells when tested in a [3H]thymidine uptake assay. Similarly, supernatants obtained from DLN/autologous tumor cocultures markedly inhibited MCA-105 proliferation; this antiproliferative effect was abrogated by the addition of anti-IFN-gamma antibody to the cultures. These results suggest that secretion of IFN-gamma by adoptively transferred DLN cells plays an essential role in tumor rejection. The dominant effect of IFN-gamma may be its demonstrated antiproliferative activity.