An important consideration in application of hepatocyte transplantation is whether the number of engrafted hepatocytes is sufficient to achieve the desired effect. Here we have evaluated the proliferative potential of transplanted primary hepatocytes during regeneration of hepatic lobes. Two million hepatocytes isolated from congeneic normal Wistar-RHA rats were injected into the main portal vein of deficient, jaundiced Gunn rats. The right branch of the portal vein was ligated 24 hr before hepatocyte transplantation (group A) or transiently clamped during hepatocyte injection (group B) or 24 hr after hepatocyte injection (group C). In these groups, the three lobes supplied by the right branch of the portal vein rapidly atrophied and disappeared in 4 days, whereas the remaining lobes proliferated, as shown by size increase and 5-bromo-2-deoxy-uridine uptake. Two control groups received 2 million (group D) or 20 million hepatocytes (group E) without ligation. Hepatocyte engraftment occurred in all groups. The greatest hypobilirubinemic effect was observed in group A, in which serum bilirubin concentrations were reduced to 1.7+/-0.45 mg/dl from pretransplantation levels of 6.9+/-1.2 mg/dl. This effect was even greater than that observed after transplantation of 20 times more hepatocytes without ligation (group E). Specific endonuclease digestion of a polymerase chain reaction-amplified segment of the ugt1 gene from hepatic DNA showed that up to 25% of the DNA was of donor origin. This paralleled the hepatic bilirubin-UDP-glucuronosyltransferase activity, which was above 50% of normal. The results indicate that the transplanted hepatocytes proliferate preferentially within the regenerating lobes, replacing more than 20% of the liver mass with the progeny of the transplanted phenotypically normal hepatocytes.