In molecular biology, type-II restriction endonuclease. which specifically cleave DNA at a limited number of sites, have been exploited as a means of characterizing DNA fragments, DNA mapping and of modifying DNA for genetic engineering. Recently, many type-II restriction endonucleases have been found to decrease their substrate specificity under modified conditions such as extreme pH, low ionic strength, high enzyme concentration, substitution of metallic cofactors, or addition of organic solvents. This study used restriction endonuclease EcoRI which are used most frequently in genetic engineering. We investigated their specificity change in buffer condition including various organic solvents. The specificity of cleavage of EcoRI is altered in the presence of hydrophobic reagents, such as ethanol, ethyleneglycol and DMSO. The enzyme recognition site was not changed randomly but by preferential order by increasing the concentration of organic solvent. When EcoRI reacted with substrate pGEM3 vector which have one canonical recognition site (GAATTC), EcoRI cleaved noncanonical TAATTC and GAGTTC subsequently in more than 10% DMSO solution. These changes of specificities depended on the hydrophobicity of organic solvent (LogP: partition coefficient). As a results, the recognition sequence site was changed in the presence of organic solvents whose LogP are -2.0~0. The specificities were not easily changed in enzyme inactivating organic solvent such as acetone, 2-methyl-2-propanol, 2-methyl-1-propanol. These results might show that restriction enzyme could be used to cleave at unusual site by changing the reaction condition.