Neither the presence nor the specific role of secretory cytokines in in vivo allograft rejection has been extensively studied. We quantitated the levels of colony-stimulating factors, tumor necrosis factor, and interleukin 1 within the rejecting allograft. BALB/c (H-2d) mice were implanted with polyurethane sponges containing either allogeneic C57BL/6 (H-2b) or syngeneic splenocytes, or splenocyte-free media. At various days postgrafting, the sponges were harvested, and the cells infiltrating the grafts were analyzed for specific antidonor cytolytic activity, while IL-1, TNF, and CSF levels were measured in the graft exudate fluid. Allogeneic grafts had significantly higher concentrations of CSF, TNF, and IL-1 than syngeneic of splenocyte-free grafts. A specific radioimmunoassay revealed that macrophage colony-stimulating factor (M-CSF) is the primary CSF produced in the grafts. Peak TNF levels preceded peak M-CSF and IL-1 levels, which coincided with the initial appearance of allospecific cytotoxic T lymphocytes. Maximal CTL activity was seen on day 13, when the levels of these cytokines had already begun to fall. Specific bioassays for multi-CSF (IL-3), granulocyte CSF, granulocyte-macrophage CSF, IL-2, and IL-4 failed to detect these cytokines in the sponge fluid at any time. We hypothesize that TNF, M-CSF, and IL-1 probably play regulatory roles in the immunologic events at the site of allograft challenge.