Abstract Polyphasic‐taxonomic studies of the past decade have shown that the Burkholderia cepacia complex (Bcc) comprises at least nine species, which share a high degree of 16S rDNA (98–100%) sequence similarity but only moderate levels of DNA‐DNA hybridization. Members of the Bcc are well known as opportunistic pathogens of plants, animals and humans but also as biocontrol and bioremediation agents. In this study intra‐, surface‐associated and extracellular proteins of B. cenocepacia H111, which was isolated from a cystic fibrosis patient, were examined by 2‐DE coupled to MALDI‐TOF MS. MS and MS/MS data were searched against a database comprising all currently available annotated proteins of genetically closely related strains. In total 642 proteins spots were successfully identified corresponding to 390 different protein species, which were classified into functional categories. The majority of these proteins could be linked to housekeeping functions in energy production, amino acid metabolism, protein folding, post‐translational modification and turnover, and translation. Noteworthy is the fact that a significant number of truly secreted and membrane proteins were identified in the extracellular and surface‐associated sub‐proteomes. This indicates that the pre‐fractionation protocol used in this study is a highly valuable strategy for unravelling the cellular location of the identified proteins.