The major seminal vesicle secreted proteins in human semen, semenogelin I and semenogelin II, interact non-covalently and via disulphide bridges to instantly form a coagulum upon ejaculation. The coagulum is liquefied after a few minutes due to the action of a prostatic serine protease, prostate-specific antigen (PSA). In contrast to rat semen, which forms an insoluble plug within minutes of expulsion, no transglutaminase-mediated cross-linking has been demonstrated in ejaculated human semen. However, we here show that semenogelin I and semenogelin II, both in seminal vesicle fluid and purified from semen, are substrates for factor XIIIa, the fibrin cross-linking transglutaminase. The cross-linking of the semenogelins, which was conformation-dependent, and the incorporation of a fluorescence-labelled amine, were visualised by SDS/PAGE and Western blot. Purified semenogelin I and semenogelin II could be cross-linked separately into complexes. Moreover, digestion of semenogelin with PSA produced fragments, some of which were cross-linked into complexes by factor XIIIa. We also found that PSA was unable to release any semenogelin fragments during exposure of the high molecular-mass complexes of cross-linked semenogelin to active PSA.