Electrocatalytic oxidation of guanines in DNA by Ru(bpy)(3)(3+/2+) (bpy= 2,2'-bipyridine) has been detected at 86 mu m-radius carbon electrodes. The electrocatalysis, which is apparent as a current enhancement of the Ru(bpy)(3)(3+/2+) couple in the presence of DNA, is dependent on the ionic strength. The ionic strength effect is due not to a change in the mediator-electrode surface interaction, but rather to a change in the mediator-DNA interaction. The small size of the electrode allows examination of the contribution of the time-independent current, which is a function of the diffusion coefficients of the electroactive species, to the total current generated by guanine oxidation by Ru(bpy)(3)(3+). The second-order rate constants from digital simulation of the experimental cyclic voltammograms collected at low ionic strength are (in terms of moles of guanine) (5.6 +/- 1.4) x 10(4) M-1 s(-1) at 5 mV/s and (8.8 +/- 2.2) x 10(4) M-1 s(-1) at 250 mV/s. At high ionic strength, the second-order rate constants for voltammograms collected at 5 mV/s or 250 mV/s are both 2 x 10(4) M-1 s(-1) Digital simulation of the time-independent current reveals that weak electrostatic association of Ru(bpy)(3)(2+) or Ru(bpy)(3)(3+) with DNA produces maximal currents, extensive association of either the 2+ or the 3+ form inhibits the electrocatalysis and depresses the catalytic currents. Therefore, Ru(bpy)(3)(2+), which binds to DNA with a weaker affinity than does Ru(bpy)(3)(3+), is a near-ideal catalyst for electrocatalytic guanine oxidation in DNA.