The indicator amino acid oxidation (IAAO) method is a novel method for determining protein requirements. Recently, the protein requirement of healthy young men was reevaluated using this method, and the currently recommended protein requirement based on nitrogen balance study was found to be deficient. Similarly, with respect to experimental animals, the protein concentration used widely in the experimental diets was assumed to be deficient. However, only a few studies have tested the IAAO method in experimental animals. In particular, there are no studies on the protein requirement of adult rats measured using this method. Therefore, we applied the IAAO method to adult rats, to determine their casein protein requirement. Male Wistar/ST rats (15-18 wk old, housed in lighting (lights on from 23:00 to 11:00) conditions) were provided with the test diet including graded casein (5, 7, 9, 13, 17, 21 and 25%) every 2 h from 11:00 to 17:00. Tracer administration of 13C-phenylalanine was performed hourly from 14:00 to 17:00. Breath 13CO2 was measured every 30 min after the first tracer administration. There were significant differences between the 13CO2 concentration of the 5% and 17% casein groups at 17:00 and 18:00 (p<0.05). The mean casein protein requirement and recommended dietary allowance (RDA) were estimated to be 5.2 g/kg BW/d and 7.0 g/kg BW/d using the mixed-effect change point regression model, respectively. Our results indicated that the recommended casein value may be slightly deficient to satisfy the protein metabolic demand of some adult rats.