Free fatty acids are important flavor compounds in cheese, where they contribute to pungent, rancid, cheese, and fruitynotes. They mainly result from the lipolytic activity of cheese micro-organisms. Propionibacterium freudenreichii, aspecies used as a ripening culture in Swiss cheese, is the main agent of Swiss cheese lipolysis, with 96% of the free fattyacids released during the ripening resulting from P. freudenreichii activity. Our aim was to identify the most probablelipolytic esterase(s) involved in cheese lipolysis by P. freudenreichii. Since cheese lipolysis mainly occurs during P.freudenreichii growth, we hypothesized that is does not result from the activity of intracellular enzymes released fromlyzed cells, and thus we focused our study on surface-exposed or secreted esterases. Out of the twelve putative esterasespreviously predicted from the genome sequence of P. freudenreichii CIRM-BIA1T, we identified one secreted esterase,PF#279, and one putative esterase predicted to be anchored in the plasma membrane, PF#774. To evaluate therespective role of these two proteins in lipolysis, P. freudenreichii CIRM-BIA1T was knocked out for the genes encoding