Peritoneal cells from C57B1/6 mice, incubated in vitro with a pure mouse granuloma protein (MGP), decreased growth of Lewis tumor cells (3LL). This cytostatic activity was due to the release of stable cytostatic factor(s) as determined by reduction of 125iododeoxyuridine (125I-UdR) incorporation in target cell cultures. Resident, inflammatory or bone-marrow-derived macrophages incubated with MGP had no cytostatic effect on tumor cells. Co-operation between lymphocytes and macrophages was required to express cytostasis when these cells were exposed to MGP. Maximal production of this factor by peritoneal cells was detected in the medium after treatment with MGP for 6 hr. Cytostatic activity was thermolabile (1 hr/56 degrees C). Fractionation of peritoneal cell supernatant by HPLC resulted in the recovery of one peak, eluting at 70 kDa, which was cytostatic for 3LL cells. The cytostatic supernatant was tested for tumor necrosis factor activity (TNF): no correlation between TNF and cytostatic activity was observed. Indeed, even high amounts of mouse rTNF did not affect 3LL cell growth. Moreover, monoclonal antibodies (MAbs) to mouse TNF had no effect on cytostasis, indicating absence of synergistic interactions of the cytostatic factor with low levels of mouse TNF.