Egg-envelope, a fibrous extracellular matrix, surrounding an oocyte is constructed from ZPC, ZPX2, both of which are secreted from ovarian granulosa cells, and ZPB1 which is secreted from liver cells and transported into ovary in birds. We report here that in vitro incubation of ZPB1 with ZPC spontaneously produced fibrous aggregates of ZPB1-ZPC hetero-complexes, which were visible under optical microscopy and morphologically resembled the aggregates obtained from mechanically decomposed chicken egg-envelope. Formation of such fibrous aggregates depended on ZPC/ZPB1 ratio, and involved ZPB1 dimerization through disulfide cross-linking, which had been found in authentic egg-envelope developed in hen's ovary. Furthermore, addition of excessive amounts of ZPC to ZPB1 produced soluble but high molecular weight hetero-complexes with increased adherence property against polystyrene ELISA plates. Thus, the specific association between ZPB1 and ZPC could play pivotal roles to initiate complex formation of hetero-polymers of ZP proteins in egg-envelope matrix construction. (C) 2007 Published by Elsevier Inc.