Whooping cough, caused by the gram negative pathogen Bordetella pertussis, is a worldwide acute respiratory disease that predominantly involves infants. In the present study, surface proteins of B. pertussis Tohama I and Saadet strains were identified by using 2DE followed by MALDI-TOF-MS/MS analysis and also geLC–MS/MS. With these approaches it was possible to identify 45 and 226 proteins, respectively. When surface proteins of the strains were separated by 2DE and analyzed by Western blotting for their reactivity, a total of 27 immunogenic spots which correspond to 11 different gene products were determined. Glutamine-binding periplasmic protein, leu/ile/val-binding protein, one putative exported protein, and iron-superoxide dismutase (Fe-SOD) were found as immunogenic for the first time in Bordetella. Of a total of 226 proteins identified, 16 were differentially expressed in B. pertussis Saadet and Tohama I strains. Five proteins were expressed only in Saadet (adhesin, chaperone protein DnaJ, fimbrial protein FimX, putative secreted protein Bsp22 and putative universal stress protein), and two (ABC transporter substrate-binding protein and a putative binding protein-dependent transport periplasmic protein) only in Tohama I.