To study on the organization and expression of eukaryotic tRNA gene, total tRNA genes of Aspergillus nidulans were cloned into E. coli. Of these clones, arginine-and aspartate-tRNA gene clone have been selected and the molecular structure of each tRNA genes has been determined. First of all, BamHl restriction endonuclease and T₄-DNA ligase were purified to establish the fundamental technique of recombinant DNA technology. The chromosomal DNA was obtained from haploid spores (conidia) by disrupting them physically and dye buoyant density gradient ultracentrifugation. pBR322 plasmid was used as a cloning vector. After in vitro recombination of Aspergillus-vector DNA using BamHl and T₄-DNA ligase, the recombinant DNAs were transformed into E. coli HB101. About 30,000 transformants were obtained from sixteen independent transformations. Among these, 5,300 colonies carrying Aspergillus DNA were selected by insertional inactivation. Colony hybridization experiments were performed with ^(32)P-labeled Aspergillus total tRNA, thus 105 clones of total tRNA gene have been selected from Aspergillus gene library. The results of clone analysis and cohybridization have revealed that tRNA genes of Aspergillus seem to be more clustered on the chromosome than those of yeast. Specific tRNAs of Aspergillus were identified either by charging test or by rapid RNA sequencing method. From charging test, the specific tRNAs such as tRNA^(Arg), tRNA^(Gly), tRNA^(Leu), tRNA^(Lys), and tRNA^(Val) were identified. With enzymatic partial digestion method using some specific RNases, tRNA^(Arg)_(ACG) and tRNA^(Asp)_(GUC) were identified and confirmed by further sequence analysis including identification of invariant bases and comparison of sequence homology. Modified bases of tRNA^(Arg) were analyzed by Silberklang`s method. As a result, dihydrouracil, 5-methyluridine, 1-methylguanosine, and 2-methylguanosine were identified but 3 spots have not yet been identified. One of which is thought to be inosine. One clone (pANt32) showed positive signal when the total tRNA gene clones were hybridized with ^(32)P-labeled tRNA^(Arg) probe, and pANt12 responded with tRNA^(Asp) probe. Restriction map of these two plasmids have been made, and each tRNA genes were localized by Southern hybridization. Nucleotide sequences of each plasmids have been determined with Maxam and Gilbert`s chemical cleavage methods. Each tRNA genes have been identified and tDNA sequences are coincide with the cognate RNA sequences. The most striking features of each tRNA gene are those that tRNA^(Asp) gene contains intervening sequences of 15 b.p. in which sequences complementary to anticodon sequences are not found and the long T-stretch is located just after the structural gene of tRNA^(Arg). In addition, the sequence homology of above two tRNAs to each tRNA family has been analyzed with computer. And the possible phylogenic relationship between species have also been deduced by comparing the nucleotide sequence homology of tRNA gene.