Various cell-surface multisubunit protein polymers, known as pili or fimbriae, have a pivotal role in the colonization of specific host tissues by many pathogenic bacteria. In contrast to Gram-negative bacteria, Gram-positive bacteria assemble pili by a distinct mechanism involving a transpeptidase called sortase. Sortase crosslinks individual pilin monomers and ultimately joins the resulting covalent polymer to the cell-wall peptidoglycan. Here we review current knowledge of this mechanism and the roles of Gram-positive pili in the colonization of specific host tissues, modulation of host immune responses and the development of bacterial biofilms.