Abstract The correct performance of wine malolactic fermentation (MLF) depends on the metabolic characteristics of the Oenococcus oeni strain/s responsible for this process. This study characterizes four O. oeni strains, which behave differently in terms of malolactic performance, the citric acid use related to acetic acid production, and stress adaptation. Metabolic evolution and its associated enzymatic activities were studied and the transcriptional response of the genes related to MLF, citrate metabolism and stress response was compared among strains. A higher initial expression of both the malolactic enzyme and the encoding gene mleA may be related to faster MLF. The initial transcriptional levels of citrate lyase ( citE) proved indicative of early citrate consumption. Moreover, the strains that performed best in wine-like conditions presented a much higher relative expression of several stress responsive genes, particularly hsp18, clpP, ctsR and rmlB. Finally, an inter-strain comparison of the transcriptional levels of selected genes at different times during MLF proved a useful tool in characterizing strains based on their metabolic behaviour.