Trauma victims may suffer from repeated hemorrhage, but responses of cytokines to it have not been described. To study this question, we first detected the time course of changes in serum tumor necrosis factor (TNF) activity and hepatic TNF- mRNA by cytotoxicity against L929 cells and by reverse transcription (RT) and polymerase chain reaction (PCR), respectively, after different sizes of hemorrhage (10-20 ml/kg) with chronically cannulated rats. Then we examined the changes in TNF- mRNA when two sequential 10 ml/kg hemorrhages were performed. TNF activity showed no significant increases after either size of hemorrhage. At mRNA level, both 15 ml/kg and 20 ml/kg hemorrhages induced significant increases after hemorrhage, whereas a 10 ml/kg hemorrhage did not. When the 10 ml/kg hemorrhage was repeated 24 h later, however, TNF- mRNA showed a significant increase. There were no significant differences in blood pressure and heart rate after single and repeated 10 ml/kg hemorrhage. This potentiation persisted for 48 h. These results show that responses of hepatic TNF- mRNA are augmented when moderate hemorrhage is repeated.