Dealkylation of diethylnitrosamine (DEN) by a liver microsomal fraction was measured after treatment of the Mongolian gerbil with DEN, Valium, or DEN plus Valium. Valium-dosed male and female animals showed strongly stimulated deethylase levels compared with the controls. This was also true of the DEN-treated females. Combined treatment (DEN + Valium) resulted in a significant loss of activity compared with treatment with DEN only, but was not significantly lower than that in the controls. Significant differences were also found between the experimental groups in microsomal enzymes. Metabolism of 14C-DEN to 14CO2 by gerbil liver slices in vitro showed dose-dependent inhibition by Valium. Kinetic analysis of DEN-dealkylation by purified gerbil microsomes revealed at least two Km values. In the microsomal system, diazepam inhibited DEN-dealkylation significantly at low substrate levels.