Butanol tolerance is a critical factor affecting the ability of microorganisms to generate economically viable quantities of butanol. Current Clostridium strains are unable to tolerate greater than 2% 1-butanol thus membrane or gas stripping technologies to actively remove butanol during fermentation are advantageous. To evaluate the potential of alternative hosts for butanol production, we screened 24 different microorganisms for their tolerance to butanol. We found that in general, a barrier to growth exists between 1% and 2% butanol and few microorganisms can tolerate 2% butanol. Strains of Escherichia coli, Zymomonas mobilis, and non-Saccharomyces yeasts were unable to surmount the 2% butanol growth barrier. Several strains of Saccharomyces cerevisiae exhibit limited growth in 2% butanol, while two strains of Lactobacillus were able to tolerate and grow in up to 3% butanol.