Normal adult human bladder obtained at cystoscopy has been maintained in long-term organ culture. Several media were tested for their ability to maintain viability and normal tissue morphology. The optimum medium was Ham's F-12 nutrient mixture, supplemented with 10% fetal calf serum, hydrocortisone (1 microgram/ml), and FeSO4, (0.45 microgram/ml). During the first 28 days in vitro, epithelial damage incurred at biopsy and during preparation of the cultures was repaired, and epithelialization of cut stromal surfaces occurred. A wave of cell proliferation was identified by [3H]thymidine autoradiography, 24-h labeling indices rising to a peak of up to 50% on the cut sides of the cultures between 7 and 21 days and falling to 0 to 5% by 21 to 28 days. The regenerating epithelium showed all the normal features of urothelial cell differentiation when examined by scanning and transmission electron microscopy. From 28 days, histology and scanning and transmission electron microscopy showed the cultured urothelium in most cultures to resemble closely that in the normal bladder in vivo, and in this mature state cultures were maintained for 100 days. Urothelium derived from certain patients, although showing normal surface maturation, developed enlarged intercellular spaces or intraepithelial mucin-containing acini. A study of the cytology of cells shed into the medium at different stages in culture showed that culture viability and epithelial differentiation could be monitored easily in long-term culture by this nondestructive means.