Aim To clone and characterize two related intracellular esterases from Oenococcus oeni and Lactobacillus hilgardii under wine-like conditions. Methods and Results The published genome sequences for O. oeni and Lact. hilgardii were used to identify, clone and purify putative esterase genes from these species designated EstCOo8 and EstC34, respectively. Both esterases are members of family V of lipolytic enzymes. However, EstC34 contains an SGSLG nucleophilic elbow structural motif instead of the usual GGSLG motif which is conserved in other lactic acid bacteria. Both esterases exhibited greatest specificity for C2C4 pNP-linked substrates and retained activity under wine-like conditions. EstCOo8 had an optimum temperature, pH, and ethanol concentration of 40 degrees C, 5.5 and 6% (v/v), respectively. Whereas EstC34 had an optimum temperature, pH and ethanol concentration of 50 degrees C, 5.0 and 10% (v/v), respectively. Conclusions Both esterases were stable and retained activity under conditions that would be encountered in wine. They have the potential to reduce short-chain ethyl esters such as ethyl acetate. Significance and Impact of the Study This study provides information that might help improve the performance of LAB during malolactic fermentation in wine in the future, either by strain selection, optimization or direct enzyme addition.