Using newly developed assay procedures, we studied the development of pyrroline-5-carboxylate synthase (PCS) and N-acetylglutamate synthase (AGAS) activity in rat tissues. PCS in the small intestine of fetuses was 1/5 that of adults and reached an adult level as early as postnatal Day 1. The highest peak was observed at Day 14, and then activity decreased to the adult level. However, PCS in the brain was highest at birth and quickly inactivated in a few days. AGAS in the fetus small intestine was 1/3 that of adults and became higher than the adult level by 40% at Day 1 but was reduced to 1/2 that of adults at Day 3. Subsequently activity increased gradually to the adult level at Day 24. On the contrary, AGAS in the fetus liver was only 1/20 that of adults, and activity increased slowly up to 10 weeks and more. Pregnancy and lactation reduced liver AGAS markedly up to Day 8 and intestinal PCS considerably up to Day 14 after parturition. PCS in the small intestine of senescent rats was almost halved compared to young controls on a whole tissue basis. AGAS in the small intestine was also halved on a gram wet weight basis. Nonetheless the liver AGAS of 430-day-old rats was higher than that of the controls, although that of 630-day rats was lower. The results indicate that the arginine synthesizing enzymes in the small intestine are highly activated in suckling and weaning, and raise a question whether arginine remains fully dispensable in pregnancy, lactation, and senescence.