Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo . It has been proposed previously that the closed (α) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys 50 and Lys 62 . In contrast, molecules in which NTP residues Lys 20 or Lys 33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys 50 and/or Lys 62 . These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.