To investigate the biological mechanism of organic solvent tolerance (OST), DNA microarrays were used to collect and compare the gene expression profiles of normal and organic solvent-tolerant Escherichia coli strains. First, we compared the tolerant-strain OST3410 to its sensitive parent strain JA300 in the absence of organic solvents. Numerous genes showed higher expression levels in OST3410, and Northern analysis was used to confirm the higher expression level of some genes. Next, the gene expression profiles of JA300 and OST3410 exposed to hexane as an organic solvent were investigated and compared with JA300 before exposure to organic solvent. In OST3410 and JA300, 115 and 47 hexane-induced genes were found, respectively. As candidates for genes related to OST, we focused on six genes: cysD, marA, mg1B, tnaA, tnaB and yihM, which were upregulated by hexane in both strains. When these genes were over-expressed on plasmids, only the marA plasmid increased OST activity. It should be noted that we succeeded in finding a gene related to OST activity using only DNA microarray data, without any biochemical or biological knowledge.