Glyoxal oxidase (GLOX) is an H2O2-producing enzyme secreted by ligninolytic cultures of Phanerochaete chrysosporium. The oxidase is reversibly inactivated during purification, but can be reactived when coupled to lignin peroxidase (LiP) with veratryl alcohol as the peroxidase substrate. To characterize the modulation of this extracellular oxidase activity, we studied effects of pH, peroxide concentration, peroxidase source (fungal vs plant), and peroxidase substrate with recombinant GLOX (rGLOX). Our results show that a peroxidase system is not required for rGLOX activity. However, the activity is transient and the enzyme is partly and reversibly inactivated by the produced peroxide. rGLOX activity is more sustained at pH 6 than pH 4.5, and therefore the activation at pH 4.5 by a coupled peroxidase system is more clearly demonstrable. Results with peroxidase substrates of widely varying redox potentials strongly suggest that oxidized intermediates produced by coupled peroxidases are the GLOX activators. Both LiP and horseradish peroxidase (HRP) may be used to fully activate rGLOX using methoxybenzenes as peroxidase substrates. Notably, rGLOX is activated when lignin itself is used in coupled reactions with Lip. In contrast, guaiacol and catechols are both inactivating and lignin degradation products are expected to have similar effects. Taken together, our results suggest that ligninolysis by peroxidase could be regulated by GLOX activity and influenced by the presence of veratryl alcohol, lignin, and lignin degradation products. Such coordinated metabolism would influence the kinetics of free radical generation by the Lips and, therefore, the overall efficiency of lignin depolymerization.