Mycoplasma hyopneumoniae is a genome-reduced pathogen which colonises the porcine respiratory tract causing a chronic pneumonia. Consequently this, in addition to the host immune response leads to reduced feed conversion within swine herds and a significant financial burden inflicted upon the industry. In order to develop successful vaccines a fundamental understanding of M. hyopneumoniae virulence mechanisms is required. Biofilm formation is one of the ways in which a number of chronic respiratory pathogens persist within their host. Here we have used time-lapse microscopy to monitor biofilm formation of M. hyopneumoniae on abiotic surfaces. M. hyopneumoniae formed prolific biofilms after prolonged incubation on a glass surface and was accelerated when cultured on a porcine epithelial cell monolayer (PK-15). In order to study the adherence of M. hyopneumoniae to these monolayers, we have used a non-hypothesis driven approach to identify those proteins which are involved in this process. Surface proteins from PK-15 monolayers were biotinylated and bound to an avidin column. This column containing the labelled surface proteins was incubated with a native M. hyopneumoniae lysate. After extensive washing, M. hyopneumoniae proteins which bound to the column were eluted in 2M NaCl and additionally in 0.4% Trifluoroacetic acid to remove any strongly bound proteins. These proteins were separated by 1D SDS-PAGE, in-gel trypsin digested and analysed by LC-MS/MS. 75% of the proteins identified have been found by our lab to reside on the M. hyopneumoniae cell surface and are deemed putative adhesins. To further examine M. hyopneumoniae adhesins we have compared the proteome of: biofilm cells, planktonic cells, and those cells which disseminate from the biofilm into the supernatant. Whole cell lysates of these 3 cell types were separated by 1D SDS-PAGE, in-gel trypsin digested and analysed by LC-MS/MS. Interestingly, two novel proteins of approximately 400 kDa each were identified in high abundance in disseminating and biofilm cells. These proteins are part of the same operon and have homology to a serine rich adhesin in another mycoplasma species. Together these proteins account for ~3% of the M. hyopneumoniae genome and thus must be integral to colonisation given the energy expenditure needed to express them. Preliminary analysis of cells containing transposon mutants in these genes appear to have a profound effect on biofilm formation. Further work will be needed to characterise these proteins and examine their role in the pathogenesis of Mycoplasma hyopneumoniae.