Neoplastic transformation of C57BL/lcrf-a' mouse bladder epithelium was induced in long-term primary cultures by a single 24-hour treatment with 7,12-dimethylbenz[a]anthracene on day 2 of culture. Transformed foci appeared earlier (40--60 days) and at a higher frequency (28%) in cultures from old donors (28--30 mo) compared with 100 days and 0.9% in cultures from young adult donors (5--7 mo). After transplantation into syngeneic mice, transformed cells produced carcinomas. Spontaneous epithelial transformation occurred in dimethyl sulfoxide-treated old cultures after the same interval (40--60 days) as in the carcinogen-treated cultures but at a lower frequency (5.3%). Spontaneous epithelial transformation did not occur in cultures from young donors.