Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for spreading of repressive chromatin. The Su(Hw) protein is responsible for activity of the best-studied Drosophila insulators. Here we demonstrate that an evolutionarily conserved protein, E(y)2/Sus1, is recruited to the Su(Hw) insulators via binding to the zinc-finger domain of Su(Hw). Partial inactivation of E(y)2 in a weak mutation, e(y)2(u1
), impairs only the barrier, but not the enhancer-blocking, activity of the Su(Hw) insulators. Whereas neither su(Hw)(-) nor e(y)2(u1
) affects fly viability, their combination proves lethal, testifying to functional interaction between Su(Hw) and E(y)2 in vivo. Apparently, different domains of Su(Hw) recruit proteins responsible for enhancer-blocking and for the barrier activity.