Medium osmolarity sensitively regulates Kupffer cell functions like phagocytosis and prostaglandin (PG) and cytokine production. Betaine and taurine, recently identified as osmolytes in liver cells, interfere with these effects. Because Kupffer cell activation is an important pathogenic mechanism in ischemia-reoxygenation injury, the influence of osmolarity and osmolytes was investigated in a rat liver perfusion model of warm ischemia. Livers were perfused with different medium osmolarities for 60 to 90 minutes in the absence of oxygen, followed by another 90 minutes of reoxygenation. Lactate dehydrogenase (LDH) leakage into the effluent perfusate during the hypoxic and the reoxygenation period was eight- to 10-fold higher with a medium osmolarity of 385 mosmol/L than in normo-osmolarity, and further decreased with hypo-osmolar perfusion buffer. Betaine and taurine addition to the perfusate in near physiological concentrations decreased hypoxia-reoxygenation-induced LDH leakage, aspartate transaminase (AST) leakage, and perfusion pressure increase in hyperosmolar and normo-osmolar perfusions. Stimulation of PGD2, PGE2, thromboxane B2 (TXB2), and tumor necrosis factor α (TNF-α) release, as well as induction of carbon uptake by the liver during reoxygenation, were suppressed by βine and taurine, pointing to an interference of these osmolytes with Kupffer cell function. In contrast, endothelial cell function as assessed by hyaluronic acid (HA) uptake was not influenced. It is concluded that warm ischemia- reoxygenation injury in rat liver is aggravated by hyperosmolarity and attenuated by hypo-osmolarity. The osmolytes βine and taurine have a protective effect, presumably by inhibition of Kupffer cell activation.