Gelatinase B or matrix metalloproteinase-9 is involved in inflammation and in autoimmune and vascular diseases. In contrast to the constitutive and homeostatic matrix metalloproteinase-2, matrix metalloproteinase-9 is an inducible enzyme. Furthermore, it needs tight regulation, and a major control mechanism of its enzymatic activity is the activation of the latent enzyme by proteolysis of the 87 residue propeptide. Activated matrix metalloproteinase-9 is detected in many vascular or hematological disease states, including in an experimental model for cerebral malaria with Plasmodium berghei ANKA. However, insight into its activation mechanism is incomplete. In view of the association with hemorrhagic and hemolytic diseases, it was studied whether and how hemoglobin and its derivatives might activate pro-matrix metalloproteinase-9. Incubation of matrix metalloproteinase-9 with hemin or beta-hematin, the core constituent of hemozoin or malaria pigment, leads to differential autocatalysis of the propeptide, mediated by allosteric interaction with the hemopexin domain. The cleavage catalyzed by beta-hematin coincides with the first cleavage by stromelysin-1/matrix metalloproteinase-3, and preincubation of matrix metalloproteinase-9 with beta-hematin enhances the activation rate by matrix metalloproteinase-3 at least 6-fold. These findings suggest that reduction of hemorrhage and hemolysis might prevent matrix metalloproteinase-9-mediated inflammatory and vascular damages.