Tn5252 is a promiscuous streptococcal element capable of madiating horizontal spread of multiple antibiotic resistance. To begin understanding the functional role of a transfer-related region in Tn5252, its nucleotide sequence was determined. Sequence of this 3. 3-kb DNA segment revealed the presence of six open reading frames. The predicted amino acid sequence of one of the open reading frames, ORF9, showed similarity to a predicted protein product of the lactococcal conjugative plasmid, pC1528. The deduced primary protein sequence of another, ORF4, showed strong structural similarity to conserved regions of various prokaryotic DNA relaxases that initiate conjugal transfer by strand- and site-specific cleavage at the transfer origin. A hybrid protein containing the ORF4 protein fused to the carboxyl terminal end of maltose binding protein was purified from Escherichia coli and found to specifically nick plasmids carrying a 2-kb DNA segment derived from the transposon. The nicking reaction is protein concentration-dependent. These results imply that the conjugative transposition of Tn5252 may involve rolling circle replication and transfer of a unique DNA strand.