Assembly in vitro of vitelline envelope (VE) components, which were precipitated by 50–70% saturated ammonium sulphate from VE extracts, was induced by the action of a sialoglycoprotein that is immunohistochemically localised in cortical alveoli of fish eggs and has serine proteinase activity. The VE components consisted of major bands of molecular mass about 150–120, 110–100, 70 and 27 kDa in addition to about 20 minor bands and contained a chorionic transglutaminase, visualised as two fluorescent bands by monodansylcadaverine staining. The VE component assembly in vitro was Ca2+-dependent, not induced if the sialoglycoprotein was pretreated with a serine proteinase inhibitor, and inhibited by the presence of p-chloromercuribenzoate, iodoacetamide or L-cysteine in the reaction medium system. Electron microscopy revealed that assembly in vitro of the VE components consisted of aggregates of network sheets, consisting of branching and anastomosing thin (approximately 27–52 nm) and thick (approximately 137–376 nm) filamentous substances. Separation by SDS-PAGE showed that a considerable number of VE components participated in the assembly in vitro in various amounts. These results suggest at least partial reproduction of the phenomena that occur in the process of fertilisation envelope (FE) formation, and provide a new approach to investigation of the process of FE assembly in vitro.