The crystal structure of the fibrinolytic enzyme tissue plasminogen activator (tPA) shows that the bulky side chain of Y99 hinders access to the active site by partially occluding the S2 site and may be responsible for the low catalytic activity of tPA toward plasminogen. We have tested the role of Y99 by replacing it with Leu, the residue found in more proficient proteases like trypsin and thrombin. The Y99L replacement results in an increase in the kcat/Km for chromogenic substrates due to enhanced diffusion into the active site. The increase is modest (threefold) for substrates specific for tPA that carry Pro or Gly at P2, but reaches 80-fold for less specific substrates carrying Arg at P2. On the other hand, the Y99L mutation has no effect on the activity of tPA toward the natural substrate plasminogen, that carries Gly at P2, and reduces more than 10-fold the inhibition of tPA by plasminogen activator inhibitor-1 (PAI-1), that carries Ala at P2. We conclude that the steric hindrance provided by Y99 in the crystal structure affects mostly nonphysiological substrates with bulky residues at P2. In addition, residue Y99 plays an active role in the recognition of PAI-1, but not plasminogen. Mutations of Y99 could therefore afford a resistance to inhibition by PAI-1 without compromising the fibrinolytic potency of tPA, a result of potential therapeutic relevance.