In order to establish whether the postnatal rise in five urea cycle enzyme activities is associated with protein synthesis, the effects of cycloheximide on urea cycle enzyme activities, citrulline and urea productions in vivo and in vitro were studied. A single injection of cycloheximide (10 μg) 9 h after birth significantly prevented the rise in enzyme activities and citrulline concentration which normally occurred after birth, while the urea level remained unchanged. These data suggest that the postnatal rise in urea cycle enzyme activities might be associated with a protein synthesis. The rate of citrulline synthesis by liver slices was reduced (about 50%) in cycloheximide-treated neonatal liver, while the rate of urea production was not significantly decreased. The results obtained in vivo are in good agreement with in vitro experiments: citrullinogenesis was only affected by cycloheximide treatment. When cycloheximide 10 mM was added to the incubation medium, the ability of control neonatal liver slices to produce citrulline and urea was reduced to 42 and 11%, respectively. Since argininosuccinic acid addition in the medium did not produce a rise in urea synthesis, it is concluded that argininosuccinase is most responsive after incubation in cycloheximide and therefore becomes the rate-limiting step of the urea synthesis.