Burkholderia pseudomallei causes septicemic melioidosis with a high rate of relapse, however microbial determinants of relapse are unknown. Proteins were analyzed from sequential B. pseudomallei isolates from primary and relapsing melioidosis. Analysis by isotope tagging for relative and absolute quantitation revealed that factors required for nitric oxide detoxification (HmpA) and necessary for anaerobic growth (ArcA, ArcC and ArcB) were highly expressed in the relapse isolate. Two-dimensional gel electrophoresis revealed up-regulation of a putative hemolysin-coregulated protein in the primary isolate, and flagellin and HSP20/alpha crystalline in the relapse isolate. These observations provide targets for further analysis of latency and virulence of melioidosis. © 2011 Institut Pasteur.