We have isolated Phe and Tyr tRNAs from Tetrahymena pyriformis mitochondria and have determined that these are "native" species, encoded by the mtDNA. A single gene for the tRNA(Phe) has been positioned 12-14 kbp from the left end of the linear Tetrahymena mtDNA, while duplicate tRNA(Tyr) genes have been localized within the inverted terminal repeats of this genome. Primary sequence analysis demonstrates that the tRNA(Tyr) has all of the characteristic primary and secondary structural features of a normal tRNA; however, the tRNA(Phe) displays several atypical features, including (i) replacement of the usual T psi sequence by UC, (ii) a U.U pair in the T psi C stem, and (iii) an extra 5'-nucleotide (U).