This paper deals with the synthesis of the 3’-half molecule of yeast alanine transfer RNA (tRNA_y~(Ala)) by ligation with T_4 RNA ligase of three component oligonucleotide fragments corresponding to nucleotides 36-45(Ⅰ), 46-57(Ⅱ) and 58-76(Ⅲ) in succession extending from the 3’-end to the 5’-end. First, in a ratio of acceptor to donor at 1.5 to 1, we adopted a method of three successive reactions, namely, the 5’-phosphorylation of the nonadecamer (Ⅲ), ligation with the dodecamer(Ⅱ) and the 5’-phosphorylation of the ligation product formed; with one isolation step and obtained the 5’-phosphorylated 31mer(46-76) (Ⅳ) in an overall yield of 70%. Then the 31met(Ⅵ) as a donor was ligated with 3 times of decamer(Ⅰ) to form the 41met(36-76) (Ⅴ), the 3’-half molecule of tRNA_y~(Ala)). The yield was 67%. After 5’-phosphorylation, (Ⅴ) was ligated with the natural 5’-half molecule to form the semi-synthetic tRNA_y~(Ala)), which was biologically active, i. e. accepting and transferring (~3H)-alanine into p This paper deals with the synthesis of the 3’-half molecule of yeast alanine transfer RNA (tRNA_y ~ (Ala)) by ligation with T_4 RNA ligase of three component oligonucleotide fragments corresponding to nucleotides 36-45 (I), 46-57 ( Ⅱ) and 58-76 (Ⅲ) in succession extending from the 3’-end to the 5’-end. First, in a ratio of acceptor to donor at 1.5 to 1, we adopted a method of three successive reactions, namely, the 5’-phosphorylation of the nonadecamer (III), ligation with the dodecamer (II) and the 5’-phosphorylation of the ligation product formed; with one isolation step and obtained the 5’-phosphorylated 31mer (46-76) ) in an overall yield of 70%. The 31met (VI) as a donor was ligated with 3 times of decamer (I) to form the 41met (36-76) (V) (Ala)). The yield was 67%. After 5’-phosphorylation, (Ⅴ) was ligated with the natural 5’-half molecule to form the semi-synthetic tRNA_y (Ala)), which was biologically active, ie accepting and trans ferring (~ 3H) -alanine into p