Alveolar type II epithelial cells have been isolated previously from rodents but not from humans. The aims of this study were to isolate and culture alveolar type II cells from human lungs, and to study their phospholipid secretion. Lung tissue was obtained from 13 patients after lobectomy or pneumonectomy. Type II cells were isolated by a modification of our method for isolating rat type II cells with porcine pancreatic elastase and discontinuous metrizamide density gradients. Cells were purified further by differential adherence and they were then cultured on an extracellular matrix prepared from bovine corneal endothelial cells. We obtained 1.3 to 4.8 X 10(6) cells per gram of tissue with cell viability on the day of isolation of 96 +/- 1% (mean +/- SE, n = 10). Type II cell purity, assessed by Papanicolaou stain on Day 1 in culture, was 84 +/- 4% (mean +/- SE) in 9 of the 13 experiments. In the remaining 4 experiments, the purity was less than 50%. Electron microscopy showed well-preserved ultrastructure. The cells, radiolabeled with 32Pi, secreted the phospholipids characteristic of pulmonary surfactant, primarily phosphatidylcholine (68%) and phosphatidylglycerol (12%). Phospholipid secretion was stimulated by tetradecanoyl phorbol acetate (4.5 times baseline) and by the beta adrenergic agonist terbutaline (2.0 times baseline), but not by the cholinergic agonist carbamylcholine chloride. In summary, human alveolar type II epithelial cells can be isolated from resected lung and maintained in culture, and they secrete the phospholipids characteristic of pulmonary surface active material.