Laccase is a copper-containing phenoloxidase, involved in lignin degradation by white rot fungi. The laccase substrate range can be extended to include nonphenolic lignin subunits in the presence of a noncatalytic cooxidant such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), with ABTS being oxidized to the stable cation radical, ABTS(+), which accumulates. In this report, we demonstrate that the ABTS(+) can be efficiently reduced back to ABTS by physiologically occurring organic acids such as oxalate, glyoxylate, and malonate. The reduction of the radical by oxalate results in the formation of H(2)O(2), indicating the formation of O(2)(-) as an intermediate. O(2)(-) itself mas shown to act as an ABTS(+) reductant. ABTS(+) reduction anal H,O, formation are strongly stimulated by the presence of Mn(2+), with accumulation of Mn(3+) being observed. Additionally, 4-methyl-O-isoeugenol, an unsaturated lignin monomer model, is capable of directly reducing ABTS(+). These data suggest several mechanisms for the reduction of ABTS(+) which would permit the effective use of ABTS as a laccase cooxidant at catalytic concentrations.