The 46-kb plasmid pSK41 is the prototype of a family of staphylococcal conjugative multiresistance plasmids. Sequence analyses have revealed the presence of a putative resolvase gene, res, on pSK41, and identical or related genes carried by other staphylococcal multiresistance plasmids. Carriage of the res region was found to ameliorate the accumulation of multimeric plasmid forms, and recombinant plasmids encoding a wild-type res gene exhibited greater plasmid segregational stability than counterparts carrying a nonfunctional mutant, irrespective of whether the cognate or a heterologous replication system and host was utilized. In vitro DNA-binding studies demonstrated that purified Res protein binds within the intergenic region upstream of the res coding sequence. Six copies of an imperfect 11-bp repeat sequence were identified within DNA sequences protected by Res in DNAseI footprinting studies, in an arrangement that suggests a typical resolution site organization consisting of three subsites.