tRNAs from fungus Aspergillus nidulans were purified by DEAE-cellulose column chromatography and polyacrylamide gel electrophoresis. These tRNAs were separated into about 40 tRNA spots on two-dimensional polyacrylamide gel electropheresis. Each isolated tRNA was eluted, aminoacylated with ³H-labeled amino acid by aminoacyltRNA synthetase which was prepared through ammonium sulfate fractionation, DEAE*cellulose and Sephadex G-50 column chromatography. We separated 4 kinds of g specific tRNAs which were going to be used as probes to find out specific tRNA clones from the total tRNA gene clones of Aspergillus. We report here that those are tRNA_(Lys), tRNA^(Gly), tRNA^(Val), and tRNA^(Leu)