The steady-state kinetics of D-glucose oxidation by ferricenium dyes RFc(+)PF(6)(-) (R = H, Me, Et, n-Bu, MeCH(2)CMe(2), and Cl) and 1,1’ - Et(2)Fc(+) PF6- catalyzed by glucose oxidase from Aspergillus niger was investigated as a function of RFc(+) and D-glucose concentrations at pH 6.7, 25 degrees C in the presence of 2% (v/v) Triton X-100. The enzymatic bleaching is characterized by large steady-state portions on the kinetic curves for all ferricenium ions studied. The reaction follows the Michaelis-Menten kinetics demonstrating a high affinity of RFc(+) toward the active site of reduced glucose oxidase (GO). The reaction rate is weakly sensitive to the nature of RFc(+), and the apparent V-m(app) values decrease only twofold on going from the most to the least reactive salt in the series (1,1’-Et(2)Fc(+) and ClFc(+), respectively), although their observed redox potentials differ by 160 mV. Remarkably, the reactivity of RFc(+) does not increase with increasing their oxidative power. The apparent Michaelis constants K-m(app) are also weakly sensitive to the nature of RFc(+). The profiles for the steady-state rate vs [HFc(+)] and [D-glucose] were rationalized in terms of the "ping-pong" mechanism typical of the catalysis by GO. Ferrocenecarboxylic acid (FcCOOH) appeared to be a competitive inhibitor of GO with the inhibition constant of (3 +/- 1) x 10-(3)M. The pH profile for the ferricenium fading is bell-shaped with the optimum around 7.5. A simple routine for a rapid in situ preparation of the ferricenium dye, which is ready for spectrophotometric assaying of the GO activity, is presented. The apparent V-m(app)and K-m(app) values for this substrate are similar to those for HFc(+) PF6-.