Occludin is a tight-junction protein controlling the integrity of endothelial and epithelial cell layers. It forms complexes with the cytoplasmic proteins ZO-1, ZO-2 and ZO-3. The ZO-binding domain in the C-terminal cytoplasmic region of human occludin has previously been isolated and identified. This domain, as expressed in a bacterial system or isolated from native cellular occludin, maintains its ability to bind ZO-1 and ZO-2. The crystallization conditions of the human ZO-binding domain are reported here. The crystals diffract to 2.3 A resolution and were shown to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 33.3, b = 35.4, c = 107.3 A.