Using a high voltage paper electrophoretic apparatus of the "Ishidai" type, free amino acids in human semen, prostatic fluid and dog prostatic fluid were separated and confirmed. Changes of amino acid composition in human semen following ejaculation were also investigated. 1) With the reference of 17 kinds of pure amino acid crystals as standards, electrophoresis with 100 volt/cm for 30 minutes confirmed 10 fractions including 2 alkaline fractions. Among the amino acids studied, overlapping occurred between tyrosin and cystine, asparagic acid and phenyl alanine, glutamic acid and methionine, leucine and iso-leucine or threonine, varine and serine, and arginine and histidine so that these were unable to separate each other. 2) As the fr ee amino acids in human semen, asparagic acic (phenyl alanine), glutamic acid, leucine (threonine) and varin (serine) were demonstrated immediately after fluctuation, followed by increased numbers in demonstrable amino acids being tyrosine (cystine), asparagic acid (phenyl alanine), glutamic acid, leucine, varine (serine), alanine, glycine, arginine and lysine. 3) Among these amino acids, particularlly large amount of asparagic acid, glutamic acid and leucine were thought to be contained in the semen. 4) The level of non-protein nitrogen in human se men was 85 mg/dl immediately after fluctuation, followed by a rapid increase with lapse of time and came to the maximum level up to 240-260 mg/dl. 5) The level of f ree amino acid in human semen was found to be about 70 mg/dl in 1 hour of ejaculation, followed by gradual increase up to the maximum of 190 mg/dl . 6) As the free amino acids in human prostatic fluid, glutamic acid , glycine, varine (serine), arginine, alanine and lysine were found. 7) In the dog prostatic fluid, glutamic acid , lcucine (threonine), varine (serine) , alanine, glycine and lysine were demonstrated. 8) No difference in co nfirmable free amino acid composition of semen between patients complaining of sterility and healthy subjects. However, a slight difference in the total level of free amino acid was demonstrated between healthy subjects and patients with aspermatogenesis.