The quinone derivative of 2,4,5-trihydroxyphenylalanine (TOPA) is a selective non-NMDA agonist and excitotoxin. While 3,4- dihydroxyphenylalanine (DOPA)-containing physiological solutions have been shown to generate TOPA and TOPA quinone (TOPA compounds), there have been no previous reports demonstrating the formation of this toxin in biological preparations. Here, using a pheochromocytoma catecholaminergic clonal cell line (PC12), we have identified TOPA compounds as by-products of catecholamine synthesis. PC12 cells incubated for 45 min with 30 microM tyrosine as a catecholamine precursor produced 1.0 +/- 0.2 pmol/10(6) cells of total TOPA compounds. The formation of these compounds could be enhanced nearly twofold when the cells were stimulated with 56 mM KCl. Moreover, the addition of a DOPA decarboxylase inhibitor (30 microM NSD-1015) increased the formation of TOPA compounds in both the unstimulated and stimulated conditions to a maximum of 5.5 +/- 0.7 pmol/10(6) cells after a 45 min incubation. A time-course analysis revealed that DOPA production above baseline levels coincided with the detectable generation of TOPA compounds. Finally, we observed an inhibition of TOPA compounds formation by 100 microM reduced glutathione, suggesting that these catecholamine products are formed from the extracellular autoxidation of DOPA. We propose that TOPA quinone may be an underestimated component of catecholamine toxicity that could be partly responsible for the demise of neurons in several neurodegenerative disorders, including Parkinson's and Huntington's disease. In addition, TOPA quinone may represent the first identified selective non-NMDA agonist that may be synthesized in the brain.