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Summary One key to the success of Pseudomonas spp. is their ability to reside in hostile environments. Pseudomonas spp. possess a cis–trans isomerase (Cti) an enzyme that converts the cis‐unsaturated fatty acids (FAs) of the membrane lipids to their trans‐isomers to rigidify the membrane and thereby resist stresses. Whereas the posttranslational Cti regulation has been previously reported, transcriptional cti regulation remains to be studied in more details. Here, we have studied cti transcriptional regulation in the solvent‐tolerant strain Pseudomonas putida F1. Two cti transcriptional start sites (cti‐279 and cti‐77) were identified with cti‐279 transcript being dominant. Expression of cti was found to increase with temperature increase, addition of the organic solvent, octanol and in the stationary growth phase. We found that cti expression was repressed by the cyclic‐AMP receptor protein (Crp) and repression required the cyclic‐AMP ligand of Crp. Production of trans‐unsaturated FAs was found to decrease after 24 h of growth. Although this decrease was accompanied by an increase in cyclopropane FA content, this was not at the expense of trans‐unsaturated FAs demonstrating the absence of competition between Cti and Cfa in FA modification.