In this study, cytokine release by tumor-draining lymph node cells sensitized in vitro (IVS-TDLN) was examined and correlated with therapeutic efficacy in adoptive immunotherapy. Mice bearing immunologically distinct MCA 207 and MCA 205 sarcoma tumors were utilized in criss-cross experiments. IVS-TDLN obtained from mice bearing 10-day subcutaneous (s.c.) tumors mediated immunologically specific regression of established 3-day pulmonary metastases, but demonstrated non-specific cytolytic reactivity against both tumors in a 4-h 51Cr-release assay. By contrast, these IVS-TDLN cells were found specifically to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN gamma) when restimulated in vitro with irradiated tumor cells. To determine the predictive value of tumor-specific cytokine release with in vivo therapeutic efficacy, a kinetic analysis of antitumor activities of TDLN obtained from animals bearing MCA 207 tumors for increasing lengths of time was performed. IVS-TDLN cells from mice bearing day-7, -10 and -14 s.c. tumors manifested tumor-specific release of GM-CSF and IFN gamma, and mediated significant antitumor reactivity in vivo. In contrast IVS-LN cells from day-0 and day-21 tumor-bearing animals did not release significant amounts of GM-CSF and IFN gamma, and were not therapeutically efficacious in vivo. Day-4 IVS-TDLN released high levels of GM-CSF and IFN gamma non-specifically, and were not therapeutic in adoptive immunotherapy at doses effective for day-7 and day-14 IVS-TDLN cells. In other experiments, IVS cells generated from different lymph node groups in animals bearing 10-day established s.c. tumors were examined and found to have unique profiles of cytokine release. In these studies, the ability of IVS cells to release specifically both cytokines as opposed to one was associated with greater therapeutic efficacy on a per cell basis. Our findings suggest that the tumor-specific releases of GM-CSF and IFN gamma are useful parameters to assess the in vivo therapeutic efficacy of immune lymphocytes.