Insulator elements were first described in Drosophila, but subsequent studies have shown that they are present in vertebrates as well (for review, see West et al. 2002). Over the past several years we have focused our attention on the properties of an insulator at the 5énd of the chicken β-globin locus that has begun to provide an understanding of how such elements function. This work, as well as studies in other laboratories, has revealed that there are two distinct kinds of insulator activities, which are different in their function. The first of these is the en-hancer-blocking activity, which can prevent interaction between a distal enhancer and a promoter when placed between them (Fig. 1A). This has the effect of preventing an incorrect interaction between regulatory elements in adjacent, but separately regulated, gene systems. The second insulator function is connected with barrier activity, which prevents condensed heterochromatin from extending into adjacent chromatin domains carrying transcrip-tionally active genes (Fig. 1B). The chicken β-globin locus extends over 30 kb. It contains four members of the globin gene family, with different programs of expression during development (Fig. 2), which have been studied extensively (Felsenfeld 1993). Strong positive regulatory elements, components of the locus control region (LCR), are distributed both upstream of the gene cluster and within it. Further upstream is a DNase I " hypersensitive site, " 5´HS4, which, unlike others in the locus, is not erythroid-specific, but is nucle-ase sensitive in all cells that have been tested (Reitman and Felsenfeld 1990). It seemed an attractive possibility that this marked the 5énd of the open chromatin domain; in fact, it was shown not long afterward that immediately upstream of 5´HS4 there is an abrupt decrease of nucle-ase sensitivity and histone acetylation in globin-expressing cells, consistent with a transition from the open chro-matin of the globin locus to a more inactive, condensed chromatin structure (Hebbes et al. 1994). We explored the possibility that this element might have the properties of an insulator (Chung et al. 1997).