Tyrosine tRNA was isolated from bovine liver and its nucleotide sequence was determined using in vitro 32p_ labeling techniques. Several important structural features of the tRNA are: the presence of gal-Q in the first position of the anticodon, acp3U at position 20, and a pair of adjacent N,N-dimethylguanosines (residues 26 and 27). A human DNA fragment harbored in a lambda phage clone was isolated, and restriction enzyme analysis revealed the presence of three tRNA genes in a 6.0-kb BamHI subfragment. Portions of the 6.0-kb DNA fragment containing the tRNA genes were sequenced by the method of Maxam and Gilbert and analyzed for transcriptional activity in vitro using homologous cytoplasmic extracts. A threonine tRNAUGU gene exhibited high transcriptional activity dependent on its 5'- flanking sequence. The enhanced transcription is not completely inhibited by alpha-amanitin. The value of studying tRNA structure in concert with the cognate tRNA. genes is discussed.