The interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn-binding proteins (FnBPs) were purified to near homogeneity using Fn-coupled Sepharose 4B-affinity column chromatography, and amino acid sequence analysis of the 45 and the 30 kDa proteins identified them as elongation factor Tu (EF-Tu) and pyruvate dehydrogenase E1 beta subunit (PDH-B) respectively. The genes for EF-Tu and PDH-B were cloned, and the entire EF-Tu gene and NH2-terminus of PDH-B (NPDH (pyruvate dehydrogenase E1 beta subunit from amino acid 1-244)-B) gene were overexpressed in Escherichia coli. The recombinant proteins, rEF-Tu and rNPDH-B, were purified to homogeneity by His-tag affinity column chromatography and used to immunize rabbits. Purified rEF-Tu and rNPDH-B bound to Fn using a ligand immunoblot assay and ELISA. Immunogold electron microscopy with polyclonal antibodies reactive against rEF-Tu (antirEF-Tu) and rNPDH-B (antirNPDH-B) and whole cell radioimmunoprecipitation (WCRIP) revealed the surface location of these proteins. Adherence of viable M. pneumoniae to immobilized Fn was inhibited by antirEF-Tu and antirNPDH-B antisera in a dose-dependent and cumulative manner. These results demonstrate that M. pneumoniae EF-Tu and PDH-B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.