The mammalian embryo is encased in a glycoproteinaceous coat, the zona pellucida (ZP) during preimplantation development. Prior to implantation, the blastocyst must undergo 'hatching' or ZP escape. In hamsters, there is a thinning of the ZP followed by a focal lysis and a complete dissolution of the ZP during blastocyst hatching. Earlier studies from our laboratory have indicated a role for cysteine proteases in the hatching phenomenon. In this study, we tested the effect of specific inhibitors of the three classes of cysteine protease on blastocyst hatching. Cystatin, an endogenous cathepsin inhibitor, blocked blastocyst hatching. Similarly, Fmoc-Tyr-Ala-diazomethane, a synthetic cathepsin inhibitor, blocked hatching. Both showed dose-dependent and temporal inhibition of hatching. However, Z-Val-Ala-Asp-fluoromethylketone, a synthetic caspase inhibitor, and calpastatin, an endogenous calpain inhibitor, had no effect on hatching. The cathepsins were localized to blastocyst cells. Exogenous addition of cathepsins L, P or B to cultured 8-cell embryos caused a complete ZP dissolution. The expression of mRNA and protein of cathepsins L and P was observed in peri-hatching blastocysts. Cathepsins L and P were detected in trophectodermal projections and in the ZP of peri-hatching blastocysts. These data provide the first evidence that blastocyst-derived cathepsins are functionally involved as zonalytic factors in the hatching of blastocysts in the golden hamster.