Ubiquitylation is a reversible protein modification that is implicated in many cellular functions. Recently, much progress has been made in the characterization of a superfamily of isopeptidases that remove ubiquitin: the deubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes). Far from being uniform in structure and function, these enzymes display a myriad of distinct mechanistic features. The small number (<100) of DUBs might at first suggest a low degree of selectivity; however, DUBs are subject to multiple layers of regulation that modulate both their activity and their specificity. Due to their wide-ranging involvement in key regulatory processes, these enzymes might provide new therapeutic targets.