Plasminogen binds with low affinity in a lysine-dependent manner to many cell types. Previously, a 54 kDa plasminogen receptor found on the surface of U-937 cells was identified as an ��-enolase-like molecule. The aims of this study were to determine whether recombinant ��-enolase (r-��-enolase), encoded by ENO1, was a plasminogen binding protein and to generate polyclonal antibodies against this antigen. Plasminogen specifically bound r-��-enolase with a K(d) 1.9 ��M and approached saturation at 10 ��M. Lysine-dependent plasminogen binding to r-��-enolase was demonstrated by a greater than 80% inhibition of binding by the lysine analogues ��-amino caproic acid and tranexamic acid, whilst only 14% inhibition occurred with the arginine analogue benzamidine. Removal of the C-terminal lysine residue of r-��-enolase with carboxypeptidase B significantly reduced its plasminogen binding capacity, suggesting that binding required C-terminal lysine residue of r-��-enolase. Binding to r-��-enolase enhanced the activation rate of plasminogen by urokinase but prevented ��2-antiplasmin from binding plasminogen. Taken together, these data suggest that the gene product of human ENO1 encodes an authentic plasminogen binding protein.