The effect of global transcriptional regulators related to carbohydrate metabolism on organic solvent tolerance (OST) in Escherichia coli has been investigated. The OSTs of the E. coli K-12 BW25113 knockout mutants acrA, acrB, cra, crp, eyaA, fnr, and m/c were investigated on the basis of colony forming efficiency on an agar plate overlaid with organic solvents. The knockout mutants of the cyclic AMP receptor protein (Delta crp) and adenylate cyclase (Delta cyaA) were found to increase their OSTs. However, their OSTs decreased to the level of the wild-type strain when the Delta cyaA cells were grown in the presence of exogenous cyclic AMP. These results indicate that the formation of the cAMP-Crp complex is related to OST. The microbial-adhesion-to-hydrocarbon test on the Delta crp and Delta cyaA mutants revealed that these mutants bound less abundantly to the organic solvent phase. In the Delta crp and Delta cyaA mutants, the expression levels of GadB and NuoG increased to the level of the wild type. The OSTs of Delta gadB and Delta nuoG mutants decreased, suggesting that the expressions of these proteins are involved in the increased OST in the Delta crp and Delta cyaA mutants.