Temperature-dependent changes in genomic DNA supercoiling might play an important role in temperature perception and responsive gene regulation. In the cyanobacterium Synechocystis, low temperatures induce the expression of the genes for fatty acid desaturases that introduce double bonds into acyl chains of lipid-bound fatty acids, thus regulating the membrane fluidity. I studied the effects of low temperature on supercoiling of the genomic DNA region that contains the regulatory elements of the desB gene for the omega3 desaturase, which is strongly induced by cold. Upon decrease in temperature, the degree of DNA supercoiling increased in this region. Novobiocin, an inhibitor of the DNA gyrase, prevented low-temperature-induced changes in DNA supercoiling and affected the expression of several desaturase genes with the most effect on desB. Decreasing in temperature induces three genes of FA desaturases encoding delta12, delta6 and omega3 desaturases in Synechocystis cells. Novobiocin inhibited completely low-temperature-induced transcription of desB, accumulation of the corresponding protein, and the formation of the omega3 unsaturated fatty acids. In the presence of novobiocin, the novobiocin-resistant mutant cells of Synechocystis responded to the low-temperature treatment in the same way as the wild-type cells in the absence of the antibiotics. Thus, temperature-induced changes in DNA supercoiling might form an essential part of a global regulation pathway leading to low-temperature acclimation in this mesophilic cyanobacterium.