Neoplastic transformation of C57BL/lcrf-a^t 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 sulfoxldetreated 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.