The mitochondrial genome of the liverwort Marchantia polymorpha does not encode the full complement of tRNAs for the threonine and isoleucine codon boxes. To find the missing tRNA genes specifically for tRNA(Thr) in mitochondria, we have searched the genomic library and identified two clones (pTT1 and pTT2), encoding the identical tRNA(Thr)(AGU) gene copy with different 5'- and 3'-flanking sequences. By northern analysis, we demonstrate considerable accumulation of the nuclear encoded tRNA(Thr) and moderate expression of native tRNA(Thr)(GGU) in mitochondria. Nonetheless, the imported and native tRNA(Thr) species together are not sufficient to translate all four threonine codons used in liverwort mitochondria, implicating mitochondrial import of at least one additional threonine isoacceptor tRNA. (C) 1997 Elsevier Science B.V.