Cultured cells of rat bladder transitional cell carcinoma line AY-27, in suspension, were assayed for galactosyl transferase (GT) by measurement of the transfer of [3H]galactose from uridine diphosphate-[3H]galactose to desialylated ovine submaxillary mucin (OSM-NANA). The assay was optimized with respect to time and to protein, uridine diphosphate galactose, OSM-NANA and Triton X-100 concentrations. This assay was then applied weekly to suspensions of exfoliated bladder cells collected from urines of rats fed the bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, and of control rats. Increases in activity over controls appeared 42 weeks after feeding the carcinogen, at a stage when bladder tumors were already microinvasive or deeply invasive, and activities at 52 weeks were about 10-fold greater than normal values. In contrast, a bladder cytotoxic agent inducing reversible hyperplasia was injected into rats, and exfoliated cells were collected from urines: these cells showed no greater GT activity than normal. Bladder tumor tissue from a transplanted tumor had the same high specific enzymatic GT activity as exfoliated cells from tumor-bearing rats.