OBJECTIVE: Lack of availability of aprotinin has resulted in increased clinical use of the alternative antifibrinolytic agents, ε-aminocaproic acid (EACA) and tranexamic acid (TXA), which are known to be associated with an increased risk of seizures. In contrast, aprotinin has previously been demonstrated to be neuroprotective through suppression of excitotoxicity-mediated neuronal degeneration via the extracellular plasminogen/plasmin system. This study compares the effect of antifibrinolytic agents on neuronal and mixed glial/neuronal cell cultures. METHODS: Mixed cortical cultures containing neuronal and glial cells were prepared from fetal mice and plated on a layer of confluent astrocytes from postnatal pups. A primary neuronal culture was obtained from the same gestational stage and plated in multiwall vessels. Slowly triggered excitotoxicity was induced by 24-hour exposure to 12.5 mM N-methyl-D-aspartate (NMDA). Apoptotic neuronal cell death was induced by exposure of primary neural cultures to 24 hours of serum deprivation. RESULTS: Compared with NMDA alone, no significant changes in cell death were observed for any dose of TXA or EACA in mixed cultures. Conversely, a clinical dose of aprotinin significantly reduced cell death by -31% on average. Aprotinin reduced apoptotic neuronal cell death from 75% to 37.3%, and to 34.1% at concentrations of 100 and 200 kIU/mL, respectively, and significantly decreased neuronal nuclear damage. These concentrations of aprotinin significantly inhibited caspase 9 and 3/7 activations; 250 kIU/mL aprotinin exerted maximal protection on primary cortical neurons. CONCLUSIONS: In contrast to aprotinin, EACA and TXA exert no protective effect against excitotoxic neuronal injury that can occur during cardiac surgery.