The intracellular distribution of glutaminyl-tRNA synthetases and their role in mitochondrial tRNA import were evaluated in the ancient eukaryote Leishmania tarentolae. The following results were obtained: (i) Glutaminyl-tRNA synthetase was detected in leishmanial mitochondria. This was unexpected because it has been postulated that, in organelles, Gln-tRNAGln is not formed by direct acylation of tRNAGln but by enzymatic transamidation of misacylated Glu-tRNAGln. (ii) Whereas the cytosolic extract is able to charge cytosolic and mitochondrial tRNAsGln, the mitochondrial matrix extract does not aminoacylate the cytosol-specific tRNAGln. This indicates that mitochondrial and cytosolic glutaminyl-tRNA synthetases are distinct. (iii) Seven of the 11 nucleotides that differ between the cytosolic and the mitochondrial tRNAGln are sufficient to convert the cytosol-specific tRNAGln into an optimal substrate for the mitochondrial enzyme. These nucleotides are arranged in three groups consisting of the nucleotides flanking the anticodon stem, the 5' nucleotide of the anticodon, and four nucleotides within the acceptor stem. And (iv), it was shown that the identity elements for recognition by the mitochondrial glutaminyl-tRNA synthetase do not overlap with a previously identified sequence segment required for mitochondrial import of the tRNAGln.