Protein profiles of Mycobacterium vanbaalenii PYR-1 grown in the presence of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) were examined by two-dimensional gel electrophoresis (2-DE). Cultures of M. vanbaalenii PYR-1 were incubated with pyrene, pyrene-4,5-quinone (PQ), phenanthrene, anthracene, and fluoranthene. Soluble cellular protein fractions were analyzed and compared, using immobilized pH gradient (IPG) strips. More than 1000 gel-separated proteins were detected using a 2-DE analysis program within the window of isoelectric point (pI) 4–7 and a molecular mass range of 10–100 kDa. We observed variations in the protein composition showing the upregulation of multiple proteins for the five PAH treatments compared with the uninduced control sample. By N-terminal sequencing or mass spectrometry, we further analyzed the proteins separated by 2-DE. Due to the lack of genome sequence information for this species, protein identification provided an analytical challenge. Several PAH-induced proteins were identified including a catalase-peroxidase, a putative monooxygenase, a dioxygenase small subunit, a small subunit of naphthalene-inducible dioxygenase, and aldehyde dehydrogenase. We also identified proteins related to carbohydrate metabolism (enolase, 6-phosphogluconate dehydrogenase, indole-3-glycerol phosphate synthase, and fumarase), DNA translation (probable elongation factor Tsf), heat shock proteins, and energy production (ATP synthase). Many proteins from M. vanbaalenii PYR-1 showed similarity with protein sequences from M. tuberculosis and M. leprae. Some proteins were detected uniquely upon exposure to a specific PAH whereas others were common to more than one PAH, which indicates that induction triggers not only specific responses but a common response in this strain.