We determined the nucleotide sequence of polymerase chain reaction products resulting from amplification of joint regions created after excision of transposons Tn5381 and Tn916 from a single site within plasmid pAD1. For both transposons, two joint sequences were observed. One (ATAGAT) was six nucleotides in length and identical to one of the junction sequences flanking the integrated transposon. This sequence also represents the original target sequence within pAD1. The other (TATGT (Tn5381) or TAGTT (Tn916)) was five nucleotides in length and identical to the junction sequence at the other end of the integrated transposons. These results suggest that excision of conjugative transposons from some insertion sites in gram-positive bacteria results in the formation of a joint region heteroduplex mismatched in nucleotide number as well as complementarity.