Co-cultures of the murine macrophagic cell line RAW 264.7 with the L929 fibrosarcoma cell line, but not with the leukemia L1210 cell line, showed enhanced NO production over control RAW 264.7 cells. This potentiating effect, which was observed in detectably mycoplasma-free conditions and required low concentrations of recombinant murine IFN-gamma, was due to soluble factors released from L929 cells and not to physical contact between the two cell types. The soluble factors were able to induce TNF-alpha in the macrophages and to potentiate the TNF-alpha release induced by IFN-gamma. Increased generation of NO in RAW 264.7 cells co-cultured with L929 cells was prevented by a neutralizing anti-TNF-alpha antibody, suggesting that TNF-alpha is an autocrine factor for iNOS expression in these conditions. Also the L929 cell line showed a 4- to 5-fold enhanced NO production following co-culture with RAW 264.7 cells, thus indicating that exposure of tumor cells to macrophages can lead to an increased iNOS expression in tumor cells themselves.