Plasmids found in six strains ofThiobacillus ferrooxidanswere mapped and compared in an effort to detect the origin of replication. Four strains yielded an identical 9.8-kb plasmid, pTFI91. Restriction mapping and Southern blot hybridization analysis were used to confirm this finding. Dissimilar plasmids found in two other strains contained a conserved 2.2-kbSacI region common to pTFI91. DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons. A comparison of the pTFI91 origin with those ofT.ferrooxidanspTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences. To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed intoEscherichia coliDH5α cells but no transformants were identified. To test the replication of pTFI91 independent of DNA polymerase I inE.coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into thepolA1mutant SF800, but chloramphenicol-resistant transformants were not detected. Electrotransformation ofT.ferrooxidansTFI-70 andPseudomonas putidaATCC 19151 also failed to yield transformants. The results suggested that the pTFI91 plasmid replicon does not function either inE.colior inP.putida. Since pTFI91 contains the same origin of replication as other plasmids in several otherT.ferrooxidansstrains, this replicon may be commonly distributed inT.ferrooxidans.Key words: nucleotide sequence, origin of replication, plasmid DNA, replicon,Thiobacillus ferrooxidans.