Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized, species-specific M. hyopneumoniae antigens. As a first step to solve these problems membrane and membrane-associated proteins were enriched from M. hyopneumoniae cells by Triton X-114 fractionation and further analyzed by 2D gel electrophoresis and Western blot analyses using convalescent sera. Two previously unknown immunogenic proteins were identified by quadrupole time-of-flight mass spectrometry and database analyses as the conserved putative lipoproteins, Mhp378 and Mhp651. Both proteins were expressed as recombinant GST fusion proteins and reacted with sera from convalescent pigs. Coated as solid-phase antigen, Mhp651 showed a distinct cross-reaction only with Mycoplasma flocculare specific rabbit hyperimmune serum, whereas Mhp378 was only recognized by the positive control serum directed against M. hyopneumoniae, thereby indicating its species specificity.