Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis ofheat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability ofthe genome se- quence, characterization ofO. oeni genes is limited, and little is known about the in vivo role ofLo18. Due to the lack ofgenetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation ofthe hsp18 gene is not presently practicable. As an alternative approach, with the goal ofunderstanding the biological function ofthe O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting ofhsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenua- tion ofhsp18 affects O. oeni survival under stress conditions. These results confirm the involvement ofLo18 in heat and acid tolerance ofO. oeni. Results ofanisotropy experiments also confirm a membrane-protective role for Lo18, as previous observa- tions had already suggested. This study describes a new, efficient tool to demonstrate the use ofantisense technology for modu- lating gene expression in O. oeni.