Insulators mark the boundaries of chromatin domains by limiting the range of action of enhancers and silencers. Although the properties of insulators have been well studied, their role in vivo has largely been a subject of speculation. Recent results make it possible to ascribe specific and essential functions to the insulators of Drosophila , yeast, and vertebrates. In some cases, insulator activity can be modulated by nearby regulatory elements, bound cofactors, or covalent modification of the DNA. Not simply passive barriers, insulators are active participants in eukaryotic gene regulation.