Administration of N-nitrosodiethylamine (diethylnitrosamine, DEN) to mice caused a loss of cytochrome P-450 and a corresponding depression in the activities of aminopyrine demethylase and aniline hydroxylase. Maximum effects were achieved 24 hr. after a single dose of 100 mg/kg. In chronic experiments, similar effects were achieved after animals had been drinking water containing 50 ppm of DEN for 12 weeks. The effects of DEN on aminopyrine demethylase could not be reproduced by collecting microsomes, from homogenates which had been treated with DEN in vitro. Homogenates prepared from livers of mice treated chronically with DEN were used to activate compounds to mutagens in the Salmonella/microsome test of Ames. Activation by these homogenates was not lower than activation by homogenates prepared from control animals. In fact, activation of aflatoxin B1 was enhanced by use of homogenates from DEN-treated animals as source of activating enzymes.