Aims The aim of this work was to clarify the mechanism of monounsaturated fatty acid (MUFA) synthesis in Bradyrhizobium TAL1000 and the effect of high temperature on this process. Methods and Results Bradyrhizobium TAL1000 was exposed to a high growth temperature and heat shock, and fatty acid composition and synthesis were tested. To determine the presence of a possible desaturase, a gene was identify and overexpressed in Escherichia coli. The desaturase expression was detected by RT-PCR and Western blotting. In B. TAL1000, an aerobic mechanism for MUFA synthesis was detected. Desaturation was decreased by high growth temperature and by heat shock. Two hours of exposure to 37 degrees C were required for the change in MUFA levels. A potential 9 desaturase gene was identified and successfully expressed in E.coli. A high growth temperature and not heat shock reduced transcript and protein desaturase levels in rhizobial strain. Conclusions In B. TAL1000, the anaerobic MUFA biosynthetic pathway is supplemented by an aerobic mechanism mediated by desaturase and is down-regulated by temperature to maintain membrane fluidity under stressful conditions. Significance and Impact of the Study This knowledge will be useful for developing strategies to improve a sustainable practice of this bacterium under stress and to enhance the bioprocess for the inoculants' manufacture.