Three series of nitrogen-balance experiments were carried out on growing rats fed on purified isonitrogenous diets (16 g N/kg) to study the importance of non-essential N and the essential:total N (E:T) ratio for attaining maximum N balance (NB) and biological value (BV) of protein. Minimum dietary levels of asparagine, proline and glutamic acid required for maximum NB and BV were estimated to be 1.0, 2.0 and 5.0 g/kg respectively. In an essential amino acid-based diet, the levels of individual amino acids were successively reduced to 110% of the requirement. Reducing the level of arginine, lysine or methionine + cystine resulted in a significant increase in NB and BV while the response of rats given the isoleucine-reduced diet significantly decreased. Addition of asparagine, proline and glutamic acid in the estimated minimum amounts to an essential amino acid-based diet resulted in a significant increase in NB and BV. A further significant increase was found when the levels of arginine, lysine and methionine + cystine in the diet were reduced to 110 % of the requirement. The performance of rats fed on the latter diet was similar to that of rats given a diet with the optimum E:T ratio. It is concluded that the optimum protein utilization may be influenced by the presence of some non-essential amino acids and by the surplus of some essential amino acids rather than by the E:T ratio per se.