The role of the cell envelope in the solvent tolerance mechanisms of Pseudomonas putida was investigated. The responses of a solvent-tolerant strain, P. putida Idaho, and a solvent-sensitive strain, P. putida MW1200, were examined in terms of phospholipid content and composition and of phospholipid biosynthetic rate following exposure to a nonmetabolizable solvent, o-xylene. Following o-xylene exposure, P. putida MW1200 exhibited a decrease in total phospholipid content. In contrast, P. putida Idaho demonstrated an increase in phospholipid content 1 to 6 h after exposure. Analysis of phospholipid biosynthesis showed P. putida Idaho to have a higher basal rate of phospholipid synthesis than MW1200. This rate increased significantly following exposure to xylene. Both strains showed little significant turnover of phospholipid in the absence of xylene. In the presence of xylene, both strains showed increased phospholipid turnover. The rate of turnover was significantly greater in P. putida Idaho than in P. putida MW1200. These results suggest that P. putida Idaho has a greater ability than the solvent-sensitive strain MW1200 to repair damaged membranes through efficient turnover and increased phospholipid biosynthesis.