The 2 mu DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 mu DNA therefore cannot be achieved, and the intracellular presence of 2 mu can only be assessed by molecular analysis of the DNA complement. In addition, 2 mu alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo site-specific recombination between the endogenous 2 mu DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 mu repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 mu plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 mu DNA plasmid. This system can be utilized to introduce 2 mu DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.