We investigated the effect of recombinant human tumor necrosis factor (rhTNF) on hydroxyl radical production by established cell lines in vitro, and its implication in the killing of tumor cells by rhTNF. During incubation of TNF sensitive mouse tumorigenic fibroblast L-M cells in the presence of rhTNF, hydroxyl radical production detected by the evolution of methane gas from dimethyl sulfoxide increased gradually, at 18 hours reached 1.8 times of that in the absence of rhTNF. This increase of hydroxyl radical production was dependent on the concentration of rhTNF. The addition of iron chelator 2,2'-bipyridine which inhibits iron catalized Fenton reaction and inhibits hydroxyl radical generation, suppressed both the increase of hydroxyl radical production and the cytotoxicity induced by rhTNF. The increase of hydroxyl radical production stimulated by rhTNF was also detected in TNF sensitive human myosarcoma derived KYM cells as well as in L-M cells, but no change was detected in TNF insensitive human embryonic lung fibroblast HEL cells. These results suggest that rhTNF induced increase of hydroxyl radical production may play a significant role in the mechanism of tumor cell killing by rhTNF.