A Gram positive bacterium of the genus Rhodococcus was isolated from a contaminated site in Sydney, Australia, for its ability to tolerate and degrade high concentrations of benzene. To identify fatty acids that may impart this Rhodococcus sp. with tolerance to toxic solvents, a benzene-sensitive strain, labeled M2b, was isolated using EMS mutagenesis. A comparative analysis of fatty acid profiles showed that strain M2b was unable to increase its saturated:unsaturated ratio of fatty acids to the level achieved by the w-t strain when both strains were challenged with benzene. This was due to M2b's increased abundance of myristic acid, and decreased abundance of oleic acid. In addition, by measuring the generalized polarization of the fluorescent membrane probe laurdan using fluorescence spectroscopy, we have shown for the first time the effects of an aromatic hydrocarbon on the membrane fluidity of a Rhodococcus sp. The fluidity of the membranes increased after only 0.5 hr of exposure to benzene, thus suggesting the partitioning of benzene within the lipid bilayer. The response of this Rhodococcus sp. to benzene may suggest a mechanism for how other microorganisms survive when toxic solvents are released within the vicinity of their environment.