Recent evidence has been demonstrated that trimeric tumour necrosis factor-alpha (TNF) itself is a channel forming molecule and incubation of TNF with TNF-sensitive tumour cell line U937 increases the plasmalemmal Na+ permeability (21). Therefore, membrane potential (Em) may play a role in the cytotoxicity mediated by TNF. Results in our study demonstrate that: 1) TNF did not induce any changes of Em of L-929 cells but gramicidin and valinomycin produced positive responses as measured by two fluorescent probes, bis-oxonol and dis-C3-(5). 2) Exogenously imposed membrane depolarization by gramicidin or high potassium buffer did not attenuate the TNF-mediated cytotoxicity in L929 cells. 3) When the Em of L-cells was clamped at K(+)-equilibrium potential, TNF showed a dose-dependent cytotoxicity (determined by cell morphology and neutral red uptake assay) which was very similar to the normal TNF cytotoxic action. Taken together, evidence in our study illustrates that membrane depolarization or hyperpolarization is not required for TNF-mediated cytotoxicity.