The formation of adhesive pili on the surface of Gram-negative bacteria has been studied in detail, whereas the pilus assembly pathways in Gram-positive bacteria remain to be characterized. Gram-positive microbes use the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins; a strategy that involves sorting signals of surface protein precursors and sortase, a transpeptidase that cleaves sorting signals and links the C-terminus of surface proteins via an amide bond to the peptidoglycan cross-bridge. Recent studies with Actinomyces naeslundii, Corynebacterium diphtheriae and Streptococcus parasanguis suggest that some sortase enzymes catalyze protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria. Arthobacter photogonimos and Ruminococcus albus appear to use different strategies for pilus assembly, consistent with the notion that Gram-positive organisms, similar to Gram-negative bacteria, have evolved multiple molecular strategies for the formation of pili on microbial surfaces.