The effects of dietary conditions on the degradation of hepatic N-acetyl-L-glutamate, an essential activator of carbamoyl phosphate synthase I [EC 6.3.4.16], were studied by injecting mice with [14C]glutamate and by following the fate of labeled N-acetyl-L-glutamate. In fasted mice, the radioactivity in N-acetyl-L-glutamate reached a maximum 10 min after injection and decreased rapidly with an apparent half-life of about 14 min. In mice fed on a 60% casein diet, the radioactivity in N-acetyl-L-glutamate increased up to 30 min and then decreased more slowly with an apparent half-life of about 60 min. The remarkably prolonged retention of the radioactivity in N-acetyl-L-glutamate in fed animals was not due to continued entry of the label into the compound but principally to an increase in the half-life of the compound. A protein-free diet failed to induce a similar response. Glucagon induced a retention of the radioactivity, though the extent was much less. When mitochondria from fasted rats were incubated at 25 degrees C, about 40% of N-acetyl-L-glutamate passed from the mitochondria to the medium in 60 min. On the other hand, the rate of efflux from the mitochondria of fed rats was comparable to that of fasted animals in spite of a 6-fold higher concentration of the compound in the mitochondria. The observations, together with previous results, indicate that N-acetyl-L-glutamate degradation is positively regulated by a modification of its efflux through mitochondrial membranes.