Staircase cyclic voltammetry (SCV) and differential pulse voltammetry on fully oxidized flavodoxin from Desulfovibrio vulgaris Hildenborough at the bare glassy carbon electrode give one redox couple at a potential of -218 mV (standard hydrogen electrode (SHE)) at pH = 7.0 with an SCV peak current proportional to the scan rate. This response is caused by flavin mononucleotide (FMN), dissociated from the protein and adsorbed onto the electrode. The midpoint potential and the pK of 6.5 are equal to the values measured with free FMN in solution. When the cationic promoter neomycin is added, one additional and diffusion controlled response is observed. The midpoint potential is -413 mV (SHE) at pH 7.0 with a redox-linked pK of 4.8 for the reduced form. The temperature dependence is -1.86 mV K-1, yielding Delta S degrees = -179 J mol(-1) K-1 and Delta H degrees = -12.4 kJ mol(-1). Although the starting material was 100% quinone, no response was observed around the midpoint potential of the quinone to semiquinone reduction of -113 mV (SHE) at pH 7.0, determined in an EPR-monitored titration with dithionite. Digital simulation shows that the peak currents of the second reduction couple approach a maximum value after a few cycles if comproportionation of fully reduced and fully oxidized flavodoxin occurs in solution and a small amount of semiquinone is either present initially or is generated by mediation of electrode-bound FMN. In the latter case the heterogeneous electron transfer rate between adsorbed FMN and flavodoxin is 6.3 X 10(-6) m s(-1). The implications of this anomalous behaviour for electrochemistry on flavin enzymes like glucose oxidase are discussed.