Transplantation of isolated hepatocytes is a promising alternative to orthotopic liver transplantation in experimental animal models with acute hepatic failure and hereditary enzyme defects. Conventional light microscopy identification of hepatocytes within recipient livers has been limited due to the inability to distinguish between donor and recipient liver cells. In this study, we labeled hepatocytes intracellularly with the fluorescent dye DiI-18 prior to selective intraportal or intrasplenic transplantation. Syngeneic LEW rat hepatocytes were isolated and 2 x 10(7) fluorescence-labeled cells were transplanted by intraportal infusion selectively into 2/3 of the recipient liver lobules to avoid lethal portal hypertension. Rats were sacrificed on postop days 1, 3, 5, 10, 20, and 40. Histological examination was performed using light and fluorescence microscopy counterstained by light green dye. The quantity of transplanted hepatocytes residing within the recipient liver was determined by FACS analysis after enzymatic digestion of the recipient liver lobules. Engrafted hepatocytes were identified in the periportal regions of transplanted liver lobules. The stained hepatocytes were retrieved up to 20 days postop using fluorescent microscopy. Using FACS analysis the number of labeled hepatocytes was found to diminish over time following transplantation from 2.1% on postop day 1 to 0.5% on day 10. Labeled hepatocytes transplanted into the spleen were retrieved in clusters up to 20 days postop (the last day of observation). Furthermore, the migration of labeled hepatocytes from spleen to liver parenchyma was observed following intrasplenic transplantation. However, after selective intraportal transplantation, only fluorescent debris was found in splenic and pulmonary tissue upon examination of various organs. This article describes the method of fluorescent labeling of rat hepatocytes and reports the feasibility and limitations of using DiI-18 as a marker.