Inbred male WF rats were castrated at 40 days of age and divided into 5 groups. Group I was given no further treatment. Groups III, IV, and V received pellet implants of 5.0 mg diethylstilbestrol (DES) concurrently with castration. At 50-55 days of age, groups II, IV, and V were given drinking water containing 5.0 mg N-nitrosobutylurea (NBU) per day for 30 days (subthreshold dose). At the termination of NBU treatment, group V further received daily sc injections of 2-bromoergocryptine (CB-154; 0.4 mg/100 g body wt) four times a week throughout the experiment. None of castrated rats or rats castrated and treated with NBU alone developed hepatic tumors (HT) and pituitary tumors (PT). Incidences of HT and PT in groups III, IV, and V were 4/9 (44%) and 7/9 (78%), 15/17 (88%) and 12/17 (71%), and 17/20 (85%) and 4/20 (20%), respectively. The treatment of DES alone resulted in the concurrent development of HT and PT in castrated male rats (group III), and further NBU treatment significantly increased the incidence of HT (group IV). CB-154 treatment did not change the incidence of HT, the number of HT per rat, and the liver weight, although it significantly reduced the incidence of PT, the pituitary weight, and the serum prolactin level in castrated male rats given DES and NBU (group V). These results indicate that DES itself had a direct carcinogenic effect on the liver; this effect was not mediated by prolactin, and NBU increased the effect of DES in this process.