Two strains of Oenococcus oeni, RS1 (which carries the plasmid pRS1) and RS2 (which carries the plasmids pRS2 and pRS3), were grown in the presence of different curing agents and at different temperatures. Sublethal temperature together with acriflavine generated all possible types of cured strains, i.e., lacking pRS1 (from strain RS1), and lacking pRS2, pRS3, or both (from strain RS2). Sublethal temperature together with acridine orange only generated cured strains lacking pRS3. These results suggest that acriflavine is a better curing agent than acridine orange for O. oeni, and that pRS3 is the most sensitive to these curing agents. We also observed spontaneous loss of pRS2 or both pRS2 and pRS3 by electroporation. The ability to cure O. oeni strains of plasmids provides a critical new tool for the genetic analysis and engineering of this commercially important bacterium.