Aurintricarboxylic acid (ATA) is an endonuclease inhibitor which has been shown to block apoptotic cell death. We have now demonstrated that ATA is also an inhibitor of the Ca(2+)-activated neutral protease (calpain), a class of cytosolic enzyme that may also be activated during apoptosis. The two major calpain isoforms (mu- and m-calpain) were both inhibited by ATA with IC50's of 22 microM and 10 microM, respectively. The autolysis of purified mu-calpain was prevented by ATA in a concentration-dependent manner. Using casein zymography, it was found that the inhibition of mu-calpain by ATA was reversible. Finally, in a fetal rat cerebrocortical culture model of excitotoxicity, pre- and post-treatment of ATA (50 microM) reduced N-methyl-D-aspartate (NMDA)-induced spectrin breakdown and neuronal death, while application of ATA concurrent to NMDA challenge alone had no effect. This pattern of protection could not be explained by simple NMDA receptor antagonism. We thus propose that the neuroprotective effect of ATA could be in part due to its ability to inhibit calpain.