Muscle biopsy and blood samples were simultaneously collected from 27 healthy subjects for determination of free amino acid (AA) in plasma, muscle and erythrocytes (RBC) by reverse-phase high performance liquid chromatography and related their concentrations to deoxyribonucleic acid (DNA), muscle alkali-soluble protein, s-albumin and s-total protein. Extra- and intracellular water distribution in muscle was estimated with a modified chloride method. The majority of the AA showed higher concentration in intracellular water (ICW) than in plasma; exceptions were the concentrations of branched-chain amino acids (BCAA), phenylalanine and tyrosine exhibiting muscle/plasma and RBC/plasma ratios of 1.0-1.4. Muscle arginine and lysine as well as RBC citrulline showed higher concentrations in younger female subjects when compared to males whereas in the three compartments all other AA were present in higher concentrations in the males than in the female subjects. The levels of valine and asparagine in muscle as well as serine, ornithine, methionine and BCAA in RBC were higher in the younger than in the elderly subjects. On the other hand, muscle arginine and plasma histidine levels were higher in the elderly than in the younger subjects. There were significant positive correlations between plasma, muscle and RBC AA concentrations for a large number of AA, specially BCAA. The RBC BCAA concentrations correlated most strongly with the ratio of alkali-soluble protein to DNA in skeletal muscle (P < 0.002) and were also significantly correlated to serum albumin and total proteins. The results presented here are proposed to be used as reference data for simultaneously comparing the AA profile in muscle, plasma and RBC in various disease conditions.