The plasmin system native to bovine milk consists of the caseinolytic serine proteinase plasmin; its inactive zymogen, plasminogen; plasminogen activators; and inhibitors. Evidence in the literature indicates that whey proteins may inhibit plasmin activity, but there is very little mention of their effect on plasminogen activators. The objective of this research was to determine the effect of both unheated and heat-denatured beta-lactoglobulin (beta-LG), alpha-lactalbumin (alpha-LA), and BSA on plasminogen activators. Plasminogen activator activity was significantly stimulated by non-heat treated and denatured alpha-LA as well as by denatured beta-LG. The stimulation effect by these whey proteins was kinetically characterized, which showed that all 3 significantly increased the rate of plasminogen activation. The stimulation effect was shown to be independent of any effect of the whey proteins on plasmin activity by testing 2 different substrates, d-Val-Leu-Lys p-nitroanilide (S-2251) and Spectrozyme PL (Spec PL), in a plasmin assay. Results using S-2251 confirmed the inhibitory effect of whey proteins on plasmin observed by several researchers. However, use of SpecPL did not suggest inhibition. Ligand binding studies showed this discrepancy to be due to significant interaction between S-2251 and the whey proteins. Overall, this study indicates that whey protein incorporation into cheese may not hinder plasmin activity and may stimulate plasminogen activation. Furthermore, the results indicate the need for careful consideration of the type of synthetic substrate chosen for model work involving whey proteins and the plasmin system.