Mammalian and hepatic aspartate transcarbamylase is inhibited by phenobarbital p-nitrophenylhydrazone in a reversible and non-competitive type with Ki values 8.45 x 10(-5) and 9.64 x 10(-5) M in the reactions toward carbamyl phosphate and aspartate, respectively. In vivo inhibition occurred in a dose-dependent manner in which less than 50% of the activity was retained. These observations suggest that this inhibitor may interfere with the in vivo regulation of this enzyme and lead to an additional biological effect of phenobarbitals.