The uptake mechanism of FITC-labeled albumin (FITC-albumin) was examined in human alveolar epithelial cell line A549. FITC-albumin uptake by A549 cells was time- and temperature-dependent, and was markedly suppressed at 4°C compared with that at 37°C. The uptake was saturable, and was mediated by a high-affinity, low-capacity system and by a low-affinity, high-capacity system. In the following experiments, we focused on the low-affinity system. FITC-albumin uptake was markedly inhibited by metabolic inhibitors and by a vacuolar H⁺-ATPase, bafilomycin A₁. The uptake was inhibited by clathrin-mediated endocytosis inhibitors (phenylarsine oxide and chlorpromazine). Potassium depletion and hypertonicity that inhibit clathrin-mediated endocytosis also decreased FITC-albumin uptake. On the other hand, caveolae-mediated endocytosis inhibitors (indomethacin and nystatin) did not affect FITC-albumin uptake. In addition, FITC-albumin uptake was inhibited by macropinocytosis inhibitors such as 5-(N-ethyl-N-isopropyl) amiloride. These results suggest that the low-affinity system of FITC-albumin uptake is mediated by endocytosis in A549 cells, predominantly via a clathrin-mediated pathway. Macropinocytosis, but not caveolae-mediated endocytosis, may also be involved. Considering our previous findings, albumin may be transported by a similar mechanism and/or pathway in rat and human alveolar epithelial cells.