In squid (Loligo bleekeri) mitochondria, the two 3'-terminal nucleotides (G72-G73) of the tRNA(Tyr) gene overlap with the two 5'-terminal nucleotides (G1-G2) of the downstream tRNA(Cys) gene. To elucidate the processing mechanism(s) of the tRNA molecules derived from this region, tRNAs were analyzed by sequencing cDNAs synthesized from circularized tRNAs. Nucleotides G1-G2 in tRNA(Cys) appeared to be without post-transcriptional conversion, whereas CCA was post-transcriptionally added to the 3'-terminus. In contrast, in the majority of tRNAs(Tyr), G72-G73 were found to be converted to A72-A73, accompanied by the CCA addition. These results indicate that a precursor of tRNA(Tyr) is processed at U71 and two adenosines are attached prior to the CCA addition. Thus, we suggest that 5' processing of the precursor tRNA dominates 3' processing and maturation of the tRNA is mediated by a polyadenylylation enzyme in the mitochondria, a scenario which is consistent with the editing process proposed in land snail mitochondria. We also obtained intermediates, such as a premature tRNA lacking CCA that terminated at U71 and one with a single adenosine attached at position 72, which support the suggested maturation process. However, although we failed to detect a tRNA(Cys) lacking G1-G2 at the 5'-terminus, we obtained cDNAs for tRNA(Tyr) with G72-G73 and the CCA terminus. This inconsistent result suggests the co-existence of another process(es) in the maturation of these tRNA molecules in squid mitochondria.