Tumor-promoting effects of phenobarbital (PB) and sodium saccharin (SS) were tested in rats pretreated with N-nitrosomethylurea (NMU) with special reference to the site of their action. Male F344 rats were initially given injections of NMU (20 mg/kg i.p.) twice a week for 4 weeks, then given basal diet containing 0.05% PB or 5% SS for the next 32 weeks, and then killed. Appropriate control studies were also done. Histological examination of whole organs of the rats showed that PB promoted thyroid carcinogenesis whereas SS did not. A significant increase in the incidences of total tumors as well as papillary adenocarcinoma of the thyroid was observed in the group given PB after NMU (p less than 0.001). The incidence of papillary adenoma with or without papillary adenocarcinomas was also high in the NMU-PB-treated group. The organ-specific promoting effect of PB in the induction of preneoplastic lesions, as demonstrated by development of gamma-glutamyl transpeptidase-positive foci in the liver and of SS in papillary or nodular hyperplasia in the urinary bladder, as reported previously, was also confirmed. The incidences of papillomas in the forestomach were similar in groups treated with NMU-PB, NMU-SS, or NMU alone. The results indicate that PB is a tumor promoter in the liver and thyroid and that SS is a tumor promoter in the urinary bladder of rats.