During the cloning of a genomic DNA encoding mitochondrial thioredoxin (TRX) from the fission yeast Schizosaccharomyces pombe, its 5' flanking sequence was involved in the high-frequency of transformation. The recombinant plasmid pYEX that was constructed in the 2 mu plasmid-derived vector pYES2 gave rise to a significant high-frequency of transformation in S. pombe, compared to the vector alone. Plasmid pYEX contains 1,090 bp 5'-flanking sequences of the TRX gene that are ahead of the open-reading frame. Similar 5'-flanking sequences, which were inserted in the lacZ fusion vector YEp357R that contained the 2 mu origin of replication, also gave a high-frequency of transformation. Dissection of the 5'-flanking sequence of the TRX gene by the HindIII restriction site showed that the 782 bp flanking sequence (5' upstream of the HindIII site) was responsible for the high-frequency of transformation by the 2 mu plasmid-derived vector DNAs. The putative sequence that is involved in the high-frequency of transformation contains a very high ratio of A-T pairs. No known functions were assigned on the sequence, which was estimated from the GenBank database.