Mechanisms of FITC-albumin uptake by alveolar type II epithelial cells were examined using cultured RLE-6TN cells. Alkaline phosphatase activity and the expression of cytokeratin 19 mRNA were detected in RLE-6TN cells, which are characteristic features of alveolar type II epithelial cells. The uptake of FITC-albumin by the cells was time- and temperature-dependent, and showed the saturation kinetics of high- and low-affinity transport systems. FITC-albumin uptake was inhibited by native albumin as well as by chemically modified albumin, and by metabolic inhibitors and bafilomycin A₁, an inhibitor of vacuolar H⁺-ATPase. Confocal laser scanning microscopic analysis after FITC-albumin uptake showed punctate localization of fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by nystatin, indomethacin, or methyl--cyclodextrin (inhibitors of caveolae-mediated endocytosis), but was inhibited by phenylarsine oxide and chlorpromazine (inhibitors of clathrin-mediated endocytosis), in a concentration-dependent manner. Uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. These results indicate that the uptake of FITC-albumin in cultured alveolar type II epithelial cells, RLE-6TN, is mediated by clathrin-mediated but not by caveolae-mediated endocytosis, and intracellular FITC-albumin is gradually degraded in lysosomes. Possible receptors involved in this endocytic system are discussed.