OBJECTIVE To transplant undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl4)-treated mice to determine their ability to differentiate into hepatocytes in the liver. METHODS CCl4, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 (n=12), 1 x 10(5) undifferentiated ES cells (0.1 mL of 1 x 10(6)/mL solution), genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice (n=12) were injected with 0.2 mL of saline twice a week, instead of CCl4, and the same amount of ES cells was transplanted into the spleens. Group 3 mice (n=6) were treated with CCl4 and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d (PD) 10, 20, and 30. RESULTS Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice (CCl4-treated) on PD 10, however, not in those untreated with CCl4 (group 2). The GFP-positive cells were also immunopositive for albumin (ALB), alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantation during the experimental period. CONCLUSIONS Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.