Amyloid-beta plaques are a pathological hallmark of Alzheimer's disease. Several proteases are known to cleave/remove amyloid-beta, including plasmin, the product of tissue plasminogen activator cleavage of the pro-enzyme plasminogen. Although plasmin levels are lower in Alzheimer brain, there has been little analysis of the plasminogen activator/plasmin system in the brains of Alzheimer patients. In this study, zymography, immunocapture, and ELISAs were utilized to show that tissue plasminogen activator activity in frontal cortex tissue of Alzheimer patients is dramatically reduced compared with age-matched controls, while tissue plasminogen activator and plasminogen protein levels are unchanged; suggesting that plasminogen activator activity is inhibited in the Alzheimer brain. Analysis of endogenous plasminogen activator inhibitors shows that while plasminogen activator inhibitor-1 and protease nexin-1 levels are unchanged, the neuroserpin levels are significantly elevated in brains of Alzheimer patients. Furthermore, elevated amounts of tissue plasminogen activator-neuroserpin complexes are seen in the Alzheimer brain, and immunohistochemical studies demonstrate that both tissue plasminogen activator and neuroserpin are associated with amyloid-beta plaques in Alzheimer brain tissue. Thus, neuroserpin inhibition of tissue plasminogen activator activity leads to reduced plasmin and may be responsible for reduced clearance of amyloid-beta in the Alzheimer disease brain. Furthermore, decreased tissue plasminogen activator activity in the Alzheimer brain may directly influence synaptic activity and impair cognitive function.