This paper deals with the synthesis of the 3'-half molecule of yeast alanine transfer RNA (tRNAAlay) by ligation with T4 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 31mer(IV) as a donor was ligated with 3 times of decamer (I) to form the 41mer(36-76) (V), the 3'-half molecule of tRNAAlay. The yield was 67%. After 5'-phosphorylation, (V) was ligated with the natural 5'-half molecule to form the semi-synthetic tRNAAlay, which was biologically active, i.e. accepting and transferring (3H)-alanine into proteins.