This paper deals with the synthesis of nonanucleotide AGDCGGDAG_p of the dihy-drouridine(D)loop of yeast alanine transfer RNA by ligation of chemically or enzymatically synthe-sized AGDCGG with excessive pDAG_p in the presence of T_4-RNA ligase and the choice of theoptimum conditions of 5’-phosphorylation and ligation of the fragments AG,AGDC,GG,GG_pand DAG which were the constituents of the named nonanucleotide.The final product,nonanu-cleoside octaphosphate,was obtained by removing 3’-terminal phosphate group of the nonanucleo-tide with polynucleotide kinase possessing 3’-phosphatase activity and was checked by the sequenceanalysis in accordance with AGDCGGDAG.It was found that the yield of 5’-phosphorylation ofDMP or DAG were almost quantitative at 5—10℃.The synthetic AGDCGGDAG_p had been used for the total synthesis of t-RNA_y~(Ala). This paper deals with the synthesis of nonanucleotide AGDCGGDAG_p of the dihy-drouridine (D) loop of yeast alanine transfer RNA by ligation of chemically or enzymatically synthe-sized AGDCGG with excessive pDAG_p in the presence of T_4-RNA ligase and the choice of the optimum conditions of 5’-phosphorylation and ligation of the fragments AG, AGDC, GG, GG_pand DAG which were the constituents of the named nonanucleotide.The final product, nonan-cleoside octaphosphate, was obtained by removing 3’-terminal phosphate group of the nonanucleo- tide with polynucleotide kinase possessing 3’-phosphatase activity and was checked by the sequence analysis in accordance with AGDCGGDAG. It was found that the yield of 5’-phosphorylation of DMP or DAG were almost quantitative at 5-10 ° C. The synthetic AGDCGGDAG_p had been used for the total synthesis of t-RNA_y ~ (Ala).