A large number of cytoplasmic tRNAs are imported into the kinetoplast-mitochondrion of Leishmania by a receptor-mediated process. To identify the sequences recognized by import receptors, mitochondria were incubated with a combinatorial RNA library. Repeated cycles of amplification of the imported sequences (SELEX) resulted in rapid selection of several import aptamers containing sequence motifs present in the anticodon arm, the D arm, the V-T region, and acceptor stem of known tRNAs, confirming or suggesting the presence of import signals in these domains. As predicted, truncated derivatives of tRNA(Ile)(UAU) containing the D arm or the V-T region were imported in vitro. Four aptamers were studied in detail. All were imported in vitro as well as in transiently transfected cells, using the same pathway as tRNA, but their individual import efficiencies were different. Two types of aptamers were discernible: the A arm and D arm homologues (type I), which were efficiently transferred across the inner mitochondrial membrane, and the V-T homologues (type II), which were not. Remarkably, subnanomolar concentrations of type I RNAs stimulated the entry of type II RNAs into the matrix, whereas type II RNAs inhibited inner membrane transfer of type I RNAs. Moreover, tRNA(Tyr)(GUA) and tRNA(Ile)(UAU) interacted with one another as type I and type II, respectively. Such cooperative and antagonistic interactions may allow the use of a limited number of receptors to recognize a large number of tRNAs of variable affinity and enable the maintenance of a properly balanced tRNA pool for mitochondrial translation.