Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Several observations suggest that the plasminogen activator/plasmin system might also play a role in mammalian fertilization. Movement characteristics of bovine sperm incubated with different concentrations of plasmin were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4h in a modified Tyrode's medium (control) and 0.1, 1, 10 and 100 mU/ml of plasmin. The percentage motile sperm was significantly higher at 0 h for sperm incubated in 1, 10, and 100 mU of plasmin. Relative to sperm incubated in control medium, lateral head displacement (ALH), curvilinear velocity, beat cross frequency, path velocity and straight line velocity (VSL) of sperm treated with 100 mU of plasmin for 0 h were increased. After 2h of incubation, sperm treated with 100 mU of plasmin showed an increase in ALH, but a decrease in VSL, straightness and linearity. The effect of plasmin on most motility parameters appears to be direct since all these parameters were affected at 0 h of incubation. Our results support the notion of hyperactivation of bovine spermatozoa following incubation with different concentrations of plasmin. The present work provides additional information to further characterize motility movement of bovine sperm associated with final preparation for fertilization.