To investigate whether genes other than the operons nahAaAbAcAdBFCED and nahGTHINLOMKJ of Pseudomonas putida are involved in the tolerance of the bacterium to naphthalene. Cellular responses of P. putida ND6 grown with 2 and 4 g naphthalene/l were investigated using a quantitative proteomic-based approach. Comparative analysis of the proteome data identified that the expression levels of 22 proteins involved in heat shock and universal stress response, naphthalene degradation, cell envelope synthesis, and motility were up-regulated; while the expression levels of 26 proteins involved in protein and fatty acid synthesis, carbon compound, nucleotide, and amino acid metabolism, and small molecule transport were down-regulated. Our findings offer insights into the cellular response of P. putida to high naphthalene concentrations at the protein level.