The metabolic fate and carcinogenicity fo three omega-hydroxy derivatives of N-alkyl-N-(4-hydroxybutyl) nitrosamines, potent bladder carcinogens, were investigated in the rat. They were N-(2-hydroxyethyl)-N-(4-hydroxybutyl)-nitrosamine (HEHBN), N-(3-hydroxypropyl)-N-(4-hydroxybutyl) nitrosamine (HPHBN), and N-N-bis(4-hydroxybutyl) nitrosamine (BHBN). The principal urinary metabolite of HEHBN as well as HPHBN was identified as the corresponding 3-carboxypropyl compound, while the main metabolite of BHBN was N,N-bis (3-carboxypropyl) nitrosamine, indicating the preferential metabolic oxidation of the 4-hydroxybutyl chain to the 3-carboxypropyl group in the N-(omega-hydroxyalkyl)-N-(4-hydroxybutyl) nitrosamines. All three N-nitrosamines having the 4-hydroxybutyl chain induced neither bladder tumor nor any tumor in other organs under conditions similar to those used for N-alkyl-N-(4-hydroxy-butyl) nitrosamines. The essential structural and metabolic requirements in N-nitrosamines for the induction of bladder cancer in the rat are discussed.