A number of N-nitrosamines are powerful respiratory tract carcinogens which require metabolic activation in the host organism. This metabolic activation is believed to be mediated by cytochrome P-450 enzymes although unequivocal evidence for this hypothesis has not yet been achieved. An experiment was conducted to investigate the effect of the P-450 enzyme inhibitor, piperonyl butoxide, on the induction of lung tumors which originate from Clara cells in hamsters treated with N-nitrosodiethylamine (DEN). The effect of piperonyl butoxide on covalent binding and distribution of the parent nitrosamine was examined in vivo after l dose of 14C-DEN. Moreover, the effect of piperonyl butoxide on the tumor incidence induced by DEN in a chronic study was investigated by histopathology. Piperonyl butoxide significantly inhibited metabolism of DEN in the respiratory tract and inhibited the induction of lung tumors. These data provide the first experimental evidence for cytochrome P-450 enzymes being a crucial factor to the metabolic activation of DEN in vivo.