Defined culture conditions for routine clonal growth of normal human adult bronchial epithelial cells have been developed. Serum and feeder cell requirements were abrogated by: (a) optimizing the calcium concentration in nutrient medium, MCDB 151; (b) supplementing with purified factors (epidermal growth factor, 5 ng/ml; insulin, 5 micrograms/ml; transferrin, 10 microgram/ml; hydrocortisone, phosphoethanolamine and ethanolamine, each at 5 x 10(-7) M; and trace elements); and (c) coating the surface of the culture dish with a mixture of fibronectin, collagen, and bovine serum albumin. Endothelial cell growth supplement (100 micrograms/ml) and retinoic acid (3 x 10(-10) M) further enchanced growth, whereas cholera toxin was nonmitogenic and serum supplementation (greater than 2%) markedly reduced the growth rate. Using the defined system, dissociated cultures of bronchial epithelial cells, obtained from more than 15 donors, have been subcultured at clonal densities with a colony forming efficiency of 3 to 4%. In addition, high density cultures have been subcultured more than five times with four to six population doublings per passage. The features of this system permit pathobiologic investigations of bronchial epithelial cells, e.g., aging, differentiation, and carcinogenesis using conditions that isolate the results from the influence of serum, feeder cells, and other undefined factors.