Nitric oxide (NO) is a short-lived pleiotropic mediator with a multitude of biologic functions. The inducible form of NO synthase (iNOS) is responsible for the discontinuous production of high amounts of NO and is important for the cytotoxic capacity of macrophages in rodents, whereas NO production by human macrophages or monocytes (MO) is under debate. Here we report that high amounts of NO are synthesized in cocultures of human MO with the human carcinoma cell line RT4 without further stimulation. Both cell types have to be viable and metabolically active for NO production. However, in contrast to reports by others, we could demonstrate that tumor cells and not MO are the producers of NO by the following findings: 1) NO release was induced in RT4 cells, but not in MO, by diluted supernatants (SN) of RT4/MO cocultures; 2) SN of MO stimulated with tumor cell membrane preparations were sufficient to induce NO release by tumor cells; and 3) NOS mRNA expression could be detected only in tumor cells, not in MO. Separating both cells by a cell-impermeable membrane resulted in NO amounts comparable to those in cocultures with direct cell contact, indicating one or more soluble NO-inducing factors. Considerable amounts of IL-1 beta and TNF-alpha were present in cocultures. IL-1 beta and TNF-alpha, mediators produced by activated MO, in combination induce NO release in RT4 cells. Blocking of TNF-alpha or IL-1 in SN inhibited NO release in RT4 cells. This indicates that IL-1 beta and TNF-alpha play prominent roles in iNOS induction by MO in RT4 tumor cells.