Because plasma amino acid (AA) concentrations are affected by many factors, in order to use plasma amino acid concentrations as a useful criterion for determination of AA requirements, it is necessary to elucidate the factors affecting plasma AA concentrations. In this study, the concentrations of free and protein bound AA in eight tissues were investigated. After feeding the experimental diet consisted of 16.6% crude protein and 2.90kcal ME/kg, 40-week-old 10 hens which laid on that day and expected to lay on the next day were injected pentobarbital sodium solution at 5h after oviposition. All feathers were removed. After then, eight tissues, plasma, liver, small intestine, oviduct (from fimbria to isthmus), M. pectoralis profundus, and a segment of M. tibialis anterior, skin, and remained fraction were collected to determine free and protein bound AA. The concentrations of free and protein bound AA were different among tissues, but the patterns of them were similar to each other. The ratios of free AA to protein bound AA were different among AA, but the pattern of them were similar to each other except a few cases.