Amperometric superoxide anion sensor electrodes were prepared by immobilizing cytochrome c (Cyt c) on COOH-terminated Au-alkanethiolate monolayers. Monolayers and mixed-monolayers of 3-mercaptopropionic acid (MPA) with the coadsorbate, 3-mercaptopropanol (MP), were constructed on the surface of Au electrodes. The electrochemical characteristics and the superoxide anion sensor activities of cytochrome c immobilized on the monolayers and mixed-monolayers of MPA were found to depend largely on the structure of the underlying alkanethiolate monolayer. While cytochrome c on a MPA monolayer, Au/MPA/Cyt c, showed a reversible redox wave at 0.08 V (vs. Ag \ AgCl \ NaCl (sat.)), cytochrome c on the mixed-monolayer of MPA and MP, Au/MPA + MP/Cyt c, showed a wave at -0.01 V. Cytochrome c immobilized on the Au-alkanethiolate layer was supposed to acquire different conformations at each of the Au/MPA/Cyt c and Au/MPA Jr MP/Cyt c electrodes, leading to a difference in the thermodynamic potential of cytochrome c. The apparent heterogeneous electron-transfer rate constant, k(het), is determined by fast-scan cyclic voltammetry to be (2.8 +/-0.4) x 10(3) s(-1) for the Au/MPA + MP/Cyt c electrode. Both Au/MPA/Cyt c and Au/MPA + MP/Cyt c electrodes show anodic currents to O-2(-) anion generated by enzymatic reaction with a response time of similar to 15 s, and the magnitude of the current response is higher at Au/MPA + MP/Cyt c relative to that at Au/MPA/Cyt c. While H2O2 did not give any current response at the Au/MPA electrode at the applied potential of 0.15 V, cytochrome c immobilized on the Au-alkanethiolate layers was found to reduce H2O2; the cathodic current recorded for the reduction of H2O2 is very much higher at Au/MPA/Cyt c than that at Au/MPA + MP/Cyt c at all the applied potentials. The O-2(-) anion sensor activity of the two electrodes was compared with that of Au/HS(CH2)(10)COOH/Cyt c. Among the cytochrome c-modified electrodes tested, Au/MPA + MP/Cyt c showed a high anodic current for O-2(-) anion and low interferences, 17 and 7%, due to the electrochemical interferents, H2O2 and uric acid, respectively.