To determine which amino acids in a high casein diet are responsible for induction of the five urea cycle enzyme activities in rat liver, we tube-fed 21 L-amino acids singly to rats over 2 days at maximum doses which did not cause toxicity. The results were compared with the 1.3- to 1.9-fold increases (units/100 g rat) obtained by tube-feeding 2 g N/kg for 2 days as casein hydrolysate. Ala (2 g N/kg), Gly /2 g N/kg), Met (0.2--0.4 g N/kg) and Cys (0.4 g N/kg) were the only amino acids which increased all five activities. Moreover, Met. Ala, Gly and casein hydrolysate in these doses increased immuno-precipitable arginase as much as they increased its activity. A combination of Met, Ala and Gly (2 g N/kg) increased all five activities more than 2 g N/kg of casein hydrolysate. Met (0.05 g N/kg) + Ala (0.08 g N/kg) + Gly (0.1 g N/kg), the amounts of these contained in 2 g N/kg of casein, increased all five enzymes in 2 days as much as this dose of casein hydrolysate. Met (0.06 g N/kg) alone increased all five activities (units/100 g rat) 1.2 to 1.4-fold over controls by increasing g liver/100 g rat. Ammonium citrate or acetate tube-feedings over 8 days at 2 g N/kg increased only AS. The keto-acid of alanine, pyruvate, or the alpha-hydroxy acid of methionine did not increase any enzyme whereas the same molar dose of their amino acids increased all five activities. Thus three amino acids of casein, Ala, Gly and especially Met, account for the enzyme adaptation of the urea cycle on a high casein diet.