P-Glycoprotein (Pgp) has been shown to mediate multidrug resistance in tumor cell lines. Overexpression of Pgp has been detected in clinical cancer samples of many histological types. The basis and biological significance of such increases in Pgp expression are not well understood. In this study, the expression of Pgp during stepwise progression to rat liver cancer was examined to investigate the possible role of Pgp in carcinogenesis. An immunohistochemical technique was used to detect Pgp at the single-cell level, in a large number of liver nodules, hepatocellular carcinoma, and in distant metastases of the carcinomas. The results showed that distinct changes in Pgp expression occurred during stepwise liver carcinogenesis and that these changes were closely associated with the microscopic anatomy of the lesions. In contrast to gamma-glutamyl transpeptidase and glutathione S-transferase-7.7, whose expression appeared to correlate with the early steps of liver carcinogenesis, Pgp expression was higher in the large hyperplastic nodules and in hepatocellular carcinomas than in the early microscopic lesions. A particularly striking finding was the consistent expression of Pgp in the lung metastases. These findings suggested that Pgp was associated with a more progressed malignant phenotype in liver carcinogenesis.