The global warming on earth has been obvious since the 1950’s. Fossil fuels have a big impact on the observed warming and it is time to replace them with more environmentally friendly fuels. Biobutanol has been proven to be a preferred substitute to fossil fuels. The yeast Saccharomyces cerevisiae is a potential butanol producer. A problem in the biobutanol production is that the product, butanol, is toxic to the producer. In this study four S. cerevisiae strains were investigated for 1- and 2-butanol tolerance with spot tests and growth measurements with different concentrations of 1- and 2-butanol.  One of the four strains, an ale yeast, showed a higher tolerance for 1- and 2-butanol. 2-butanol was overall more tolerated by the yeast. The gene expression for the genes TMC1, LPL1, FLR1 and RPN4 was also investigated at exposure of 3 % 2-butanol. RPN4 is important in the proteasome protein degradation, which is associated with butanol tolerance. TMC1, LPL1 and FLR1 are associated to RPN4, which make them potential genes coupled to butanol tolerance. The genes TMC1 and RPN4 showed an up-regulation when exposed to 3 % 2-butanol. In conclusion, 2-butanol is preferred as a biofuel produced by ale yeast and the ideal genes to use in genetic engineering to achieve a higher butanol tolerance is TMC1 and RPN4. These results contribute to the development of an effective production of biobutanol by S. cerevisiae.