Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) is a pathogen of ruminants, causing paratuberculosis (characterized by severe emaciation). The disease is endemic in many countries including the UK and places a severe economic burden on the global livestock industry. Two types of M. a. paratuberculosis can be classified by pulsed-field electrophoresis (I/III and II), which are phenotypically distinct and appear to have different host preferences. Proteomes of Type I and Type II M. a. paratuberculosis were analyzed by 2-D gel electrophoresis to determine if any significant differences existed between the subtypes. Seven different strains of Type I and 18 strains of Type II were analyzed and compared to detect type-specific differences. These 'type-specific' differences existed regardless of growth phase and were also exhibited in cells isolated directly from pathogenic lesions. Twenty-three spots predominated on the Type I profile, from which 17 proteins were identified. Twenty-one spots predominated on the Type II profile, from which 16 proteins were identified. None of the proteins identified as differentially represented on the profiles of Type I or Type II corresponded to open reading frames of the defining genomic regions as previously described for the Type I (sheep) and Type II (cattle). Sequence polymorphisms existing in Type I and II strains were identified in some open reading frames or regulatory regions of genes that correspond to proteins expressed in a type-specific fashion. The consequence of these is discussed in relation to protein expression and their impact on the type phenotype is discussed.