Aims: Esters of short-chain fatty acids and alcohols participate significantly in the overall flavour of foods. The capacity of the lactic acid bacterium Lactococcus lactis to synthesize such esters is known even though the enzymes involved in the process are not well identified. The objective of our work is to determine whether the esterase is responsible for the whole capacity of L. lactis to synthesize esters in vitro. Methods and Results: A negative mutant for the esterase was constructed and its capacity to synthesize short chain fatty acid esters from different substrate couples was compared to that of the wild type. We observed that the esterase is responsible for the main ester synthesis activity of L. lactis in vitro. However, in the presence of some substrates, the esterase negative mutant still synthesizes low amounts of esters. Conclusions: In favourable environmental conditions, the L. lactis esterase is responsible for the main ester synthesizing activity, even though another pathway for ester synthesis probably exists. Significance and Impact of the Study: Since esters are potent aroma compounds, esterase is probably a key enzyme in the development of food flavour.