Gram-positive bacteria pose a serious healthcare threat. The growing antibiotic resistance epidemic creates a dire need for new antibiotic targets. The sortase family of enzymes is a promising target for antimicrobial therapy. This review covers the current knowledge of the mechanism, substrate specificity, and inhibitory studies of the Gram-positive bacter a enzyme sortase. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 385–396, 2010.