The egg envelope, referred to as zona pellucida (ZP) in mammalian eggs, is a fibrous and noncollagenous extracellular matrix surrounding vertebrate eggs, and composed of three to four homologous glycoproteins with a common ZP domain. In birds, a liver-derived ZP glycoprotein (ZP1/ZPB1) is transported through the bloodstream to ovarian follicles and joins the egg-envelope matrix construction together with the other ZP glycoproteins, such as ZPC and ZPD/ZPX2, both secreted from follicular granulosa cells. We report here that, through its ZP domain, ZPB1 specifically associates with ZPC, which might lead to the construction of egg-envelope matrix. The ZPB1 in laying hen's serum specifically bound to ZPC, but not to ZPX2, separated by SDS-PAGE and blotted on a membrane. Hemagglutinin (HA)-tagged ZPC expressed in a mammalian cell line (COS-7) cells was processed and secreted as a mature-form into the culture medium. From the culture supernatant of ZPC-expressing transfectants cultured in the presence of ZPB1, both ZPB1 and ZPC were recovered as heterocomplexes by immunoprecipitation using either anti-HA or anti-ZPB1 antibody. Interestingly, a monoclonal antibody, 8E1, which immunoprecipitated free ZPB1, did not immunoprecipitate the ZPB1-ZPC heterocomplexes. An 8E1 epitope was mapped on a C-terminal region of the ZP domain in a ZPB1 molecule by identifying an 8E1-positive peptide using mass spectroscopy. Furthermore, by laser scanning confocal microscopy, ZPB1 and ZPC were observed to colocalize on the surface of ZPC-expressing transfectants cultured in the presence of ZPB1, whereas almost no ZPC was detected on the surface of the transfectants cultured in the absence of ZPB1. Taken together, these results suggest that ZPB1 transported into ovarian follicles encounters and associates with ZPC secreted from granulosa cells, resulting in the formation of heterocomplexes around an oocyte. In addition, it appears that such ZPB1-ZPC complexes accumulated on the oocyte surface act as a scaffold for subsequent matrix construction events including ZPX2 association.