Tn4451 is the paradigm element of a family of mobilizable chloramphenicol resistance transposons from Clostridium perfringens and Clostridium difficile. The unique feature of these 6.3 kb elements is that their excision to form a circular molecule is mediated by TnpX, a member of the large resolvase family of site-specific recombinases. By optimizing the transposition assay system in Escherichia coli, we showed that Tn4453a from C. difficile transposed at a higher frequency than the C. perfringens element, Tn4451, and that transposition of both Tn4451 and Tn4453a was significantly enhanced by the provision of a multicopy tnpX gene in trans. The complete nucleotide sequence of Tn4453a was determined, but its comparison with Tn4451 did not reveal why it transposed at a higher frequency. Using experiments involving a chromosomal derivative of Tn4453a, we have confirmed that the circular form is the transposition intermediate. As the tnpX gene is located very close to one end of these elements, primer extension analysis was used to determine the transcription start point. The results showed that the formation of the circular intermediate creates a strong tnpX promoter, which consists of a -10 box originally located at the left end of the transposon and a -35 box originally located at the right end. The data provide strong evidence that transcription of tnpX is likely to occur from the non-replicating circular intermediate, which would facilitate the subsequent insertion of the transient circular molecule. It is postulated that, when the transposon is in an integrated state, transcription of tnpX would depend on the presence of an appropriately spaced -35 sequence in the DNA flanking the insertion site or the presence of an alternative upstream promoter.