Increasing multidrug resistance in Enterococcus faecalis, a nosocomial opportunist and common cause of bacterial endocarditis, emphasizes the need for alternative therapeutic approaches such as immunotherapy or immunoprophylaxis. In an earlier study, we demonstrated the presence of antibodies in E. faecalis endocarditis patient sera to recombinant forms of 9 E. faecalis cell wall-anchored proteins; of these, we have now characterized an in vivo-expressed locus of 3 genes and an associated sortase gene (encoding sortase C; SrtC). Here, using mutation analyses and complementation, we demonstrated that both the ebp (encoding endocarditis and biofilm-associated pili) operon and srtC are important for biofilm production of E. faecalis strain OG1RF. In addition, immunogold electron microscopy using antisera against EbpA-EbpC proteins as well as patient serum demonstrated that E. faecalis produces pleomorphic surface pili. Assembly of pili and their cell wall attachment appeared to occur via a mechanism of cross-linking of the Ebp proteins by the designated SrtC. Importantly, a nonpiliated, allelic replacement mutant was significantly attenuated in an endocarditis model. These biologically important surface pili, which are antigenic in humans during endocarditis and encoded by a ubiquitous E. faecalis operon, may be a useful immunotarget for studies aimed at prevention and/or treatment of this pathogen.