Besides its fibrinolytic function, the plasminogen-plasmin (PLG-PLA) system is also involved in fertilisation, where plasminogen activators bind to plasminogen to produce plasmin, which modulates sperm binding to the zona pellucida. However, controversy exists, depending on the species, concerning the role of the different components of the system. This study focused its attention on the role of the PLG-PLA system on fertilisation in the mouse with special attention to tissue plasminogen activator (tPA). The presence of exogenous plasminogen reduced invitro fertilisation (IVF) rates and this decline was attenuated by the presence of plasmin inhibitors in combination with plasminogen. The incubation of spermatozoa with either oocytes or cumulus cells together with plasminogen did not change the acrosome reaction but reduced the number of spermatozoa attached. When spermatozoa from tPA-/- mice were used, the IVF rate decreased drastically, although the addition of exogenous tPA during gamete co-incubation under invitro conditions increased fertilisation success. Moreover, fertility could not be restored after invivo insemination of tPA-/- spermatozoa in the female ampulla, although tPA-/- males were able to fertilise invivo. This study suggests a regulatory role of the PLG-PLA system during fertilisation in the mouse with possible implications in human reproduction clinics, such as failures in tPA production, which could be partially resolved by the addition of exogenous tPA during IVF treatment.