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Proliferation of type II cells is important for the recovery of the alveolar epithelium after acute lung injury. However, the factors that regulate the proliferation of human type II cells are unknown. Human alveolar type II cells were isolated from resected lung by dissociation with porcine pancreatic elastase and crystalline trypsin and purified by density-gradient centrifugation and serial differential adherence. The purity of the type II cells in the final adherent preparation was 84.4 ± 1.1% type II cells by alkaline phosphatase and 87.7 ± 2.8% by cytokeratin (n = 7). The medium MCDB-151 with 0.4% fetal bovine serum (FBS) was used to demonstrate the stimulatory effect of individual growth factors. Under these conditions, thymidine incorporation was stimulated by insulin, epidermal growth factor, endothelial cell growth supplement (ECGS), and acidic and basic fibroblast growth factors. Cholera toxin did not stimulate thymidine incorporation. The most effective stimulation was by the combination of insulin and ECGS. The incorporation of bromodeoxyuridine was used to identify the proportion of cells that were active in DNA synthesis. Insulin and ECGS increased the percentage of cells that incorporated bromodeoxyuridine from 8.5 ± 1.3% to 21.3 ± 2.4% (n = 6). Mitotic figures were seen in smears prepared from cultures incubated with insulin and ECGS. This observation was confirmed by electron microscopy, which demonstrated type II cells in metaphase. Increasing the concentration of FBS or human serum in the culture medium to 10% decreased the stimulatory effect of insulin and ECGS. The stimulation of thymidine incorporation by insulin and ECGS was also less when the cells were cultured on an extracellular matrix prepared from bovine corneal endothelial cells. These results are different from previous observations with rat alveolar type II cells in vitro and indicate the need for evaluating human as well as rodent type II cells for understanding the functions of this cell type. We conclude that DNA synthesis by human type II cells can be stimulated by insulin and ECGS, a mixture of heparin-binding growth factors derived from bovine neural tissue.