We studied the inhibitory effect of silibinin on ochratoxin A (OTA) and LPS-mediated tumor necrosis factor alpha (TNF-alpha) release and the leakage of cytotoxic markers glutamate dehydrogenase (GLDH) and lactate dehydrogenase (LDH), from isolated blood-free perfused rat livers, and from isolated pure rat Kupffer cells. In the recirculation perfusion model at the end point 90 min, 2.5 micromol/L OTA released 2600 pg/mL TNF-alpha without effects on liver vitality. LPS at 0.1 microg/mL induced 3000 pg TNF-alpha/mL with slight leakage of GLDH and LDH. Under similar experimental conditions, the addition of silibinin 10 min prior to OTA and LPS showed dose-dependent protection against OTA or LPS-induced hepatic TNF-alpha release. High-dose of silibinin (12.5 microg/mL) also completely restored GLDH and LDH levels in the perfusate. Pretreatment of isolated Kupffer cells with 0.02, 0.1, 0.5, 2.5, and 12.5 microg silibinin/mL 30 min prior to OTA reduced OTA-induced TNF-alpha levels to 90, 70, 25, 25, and 25% at 4 h, respectively, and abrogated any TNF-alpha release at 24 h. Similarly, in the presence of silibinin LPS-induced TNF-alpha levels decreased at 4 h to 71, 57, 18, 22, and 18%, respectively. However, after 24 h of LPS exposition the protection by silibinin vanished and TNF-alpha partially recurred into the incubation medium under LPS. In summary, silibinin had hepatoprotective effects against OTA- or LPS-mediated TNF-alpha release and also reduced the cytotoxicity of both toxins. Isolated Kupffer cells were even more sensitive to the protective effect than perfused livers and responded to very low concentrations of silibinin with a strong inhibition of toxins-mediated TNF-alpha release.