Background/aims: Conventional slice rotation culture has employed fibrin that is produced from fibrinogen by thrombin, to fix tissue slices on a slide glass. However, thrombin transmits various stimuli to cells through the thrombin receptor and affects experimental results. To exclude this disadvantage of thrombin, we developed a new holder and studied long-term liver rotation culture without using thrombin. Methods: Liver slices about [Formula: see text] were produced from the liver of 8-day-old Wister rats. The slice was fixed to a newly development holder on a slide glass and cultured in tube on rotary culture system. To evaluate whether slice survives and maintains functions, morphological structure, LDH leakage into the medium, ATP synthesis, glutathione-S-transferase activity and glycogen synthesis were examined. We also studied collagen synthesis after treatment with TGF-beta or thrombin. Results: Slice tissue survived and maintained its functions for over 10 days. After treatment with TGF-beta or thrombin, the tissue became shrunken and showed increased collagen synthesis. On the other hand, no stimulation of collagen synthesis was found in cultured tissue without treatment. Conclusions: Our data indicate that rotation culture with the new holder is suitable for long-term culture for study of liver fibrosis.