Pseudomonas putida DOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM) p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5'phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with ¹⁴C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted the exbD gene, which forms part of the exbBD tonB operon. Complementation by the exbBD and tonB genes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.