Previous studies have shown that mouse sperm bind to hamster eggs and hamster sperm bind to mouse eggs in vitro. Furthermore, sperm receptor glycoprotein isolated from the zona pellucida of unfertilised hamster (hZP3) and mouse (mZP3) eggs binds to sperm from the heterologous species. Here, we expressed the hZP3 gene, under control of a constitutive promoter (pgk-1), in mouse embryonal carcinoma (EC) cells and Chinese hamster ovary (CHO) cells stably transfected with the hZP3 gene. In both cases, recombinant hZP3 (EC-hZP3 and CHO-hZP3) secreted into the culture medium was partially purified by high-performance liquid chromatography on a size-exclusion column and assayed for bioactivity using mouse and hamster gametes. Unlike hamster egg hZP3, which binds to both mouse and hamster sperm, EC-hZP3 and CHO-hZP3 exhibits species-specific binding to hamster sperm and induce hamster sperm, but not mouse sperm, to undergo the acrosome reaction in vitro. These results provide further evidence that species-specific binding of sperm to eggs in mammals is carbohydrate-mediated. Furthermore, the results suggest that recombinant forms of mammalian sperm receptors may be useful in assessing the molecular basis of species-specific fertilisation in mammals.