In our attempt to understand the cold shock response of Bacillus subtilis, we report on the role of the B. subtilis fatty acid desaturase (FA-D) Des during membrane adaptation to low temperatures and demonstrate its importance during cold shock. A des null mutant was constructed and analysed in comparison with its parental strain. Growth studies and large-scale comparative fatty acid (FA) analysis revealed a severe cold-sensitive phenotype of the des deletion mutant during the absence of isoleucine and showed that four unsaturated fatty acid (UFA) species differing in length, branching pattern and position of the double bond are synthesized in B. subtilis JH642 but not in the des null mutant. Apart from the lack of UFA synthesis, the FA-D deletion strain showed a dramatically altered saturated fatty acid (SFA) profile at the onset of the stationary growth phase in the presence of exogenous isoleucine sources. Expression of des integrated in trans at the amyE locus of the des deletion strain not only cured the cold-sensitive phenotype observed for the des mutant but allowed much better growth than in strain JH642 after a shift from 37 degrees C to 15 degrees C. These results show that, during cold shock adaptation, des expression can completely replace the isoleucine-dependent, long-term, FA branching adaptation mechanism. We conclude that the crucial aspect in cold adaptation of the cytoplasmic membrane is not its specific molecular composition but rather its physical status in terms of its fluidity.