Aims: To advance the understanding of the molecular mechanisms underlying the capacity of bifidobacteria and lactic bacteria to convert linoleic acid (LA) into conjugated linoleic acid (CLA) by the linoleate isomerase (LI). Methods and Results: The potential LI enzymes of selected Lactobacillus, Bifidobacterium and Leuconostoc strains were compared at the genetic, amino acid sequence and functional levels. Genetic analysis was achieved by insertional mutagenesis and hybridization studies using a Lact. reuteri LI probe. Biotransformation studies monitored by gas chromatography showed that Bif. breve is a major CLA producer after the reference Lact. reuteri strain. The putative Bif. breve LI gene was PCR isolated and sequenced. Conclusions: The putative LI gene identified in this study seems essential for bacterial growth. Comparative studies indicate that the deduced protein is membrane bound and reveal the presence of several highly conserved domains among a wide range of Gram-positive bacteria. Significance and Impact of the Study: Given the multiple health benefits of CLA, the capability of some bacteria to convert LA into CLA is of great relevance. Nevertheless, the yields of CLA remain low, and the regulation of the process is far from being understood. A deeper knowledge of this capacity by the genetic studies is revealing the identity of the LI and will eventually contribute to its control.