Trefoil domains are protein structures characterized by six cysteines, which form a three-loop structure by disulfide bridges. Between the loops, a hydrophobic groove builds a binding pocket for carbohydrates or proteins. The porcine zona pellucida protein ZP4 contains such a trefoil domain (TFD); therefore we suggested a potential sperm receptor function. Sperm-egg interaction is known to be a multistep event; thus the aim of the study was to analyze the interaction of porcine TFD with boar sperm cells at several levels which reflect the most important steps of the fertilization cascade. We tested by Far Western blot and surface plasmon resonance whether a synthetic porcine TFD (pTFD) was able to interact with sperm cell lysates and by immunocytochemistry if it is able to bind sperm cell membranes. No interaction could be shown, although we previously demonstrated the threedimensional folding of the synthetic pTFD by a trypsin resistance assay. Furthermore, we determined the ability of pTFD to affect sperm capacitation or to induce the acrosome reaction. An influence was not measurable for capacitation or for the induction of acrosome reaction. We also investigated the influence of porcine TFD on sperm-ZP binding. In contrast to an anti-pZP antibody, the pTFD was not able to inhibit sperm binding to oocytes. In summary, the sperm receptor function of TFD within pig zona pellucida could not be proven. Further research is necessary to identify the function of TFD within zona pellucida; a structural role during oviduct passage is suggested.