Anomalous peak broadening is a persistent feature observed in the cyclic voltammetry of cytochrome c (cyt c) adsorbed on COOH-alkylthiolate/Au self-assembled monolayer electrodes. The origins of this broadening have been explored through variation of the electroactive cytochrome c surface concentration (Gamma). Cytochrome submonolayers were prepared using two different methods. In the first method, submonolayers were created by partial desorption of an electroactive cyt c monolayer by exposure to higher ionic strength buffers. The apparent voltammetric properties of these submonolayers were dependent on Gamma, exhibiting decreasing formal potentials, increasing electron-transfer rates, and decreasing peak widths as Gamma --> 0. On the other hand, submonolayers prepared according to the second method, in which partial desorption of a preadsorbed blocking monolayer of electroinactive porphyrin cyt c was followed by adsorption of active cyt c to the unblocked sites, exhibited opposite trends, i.e., increasing formal potentials and. decreasing electron-transfer rate as Gamma --> 1. These results are indicative of a heterogeneous population of cyt c adsorbates on the SAM/Au surface due to a distribution of interfacial environments and adsorption energies. Peak broadening under reversible electrochemical conditions has been specifically attributed to a thermodynamic distribution of cyt c differential adsorption energies (i.e., ferri- vs ferro-cyt c). Fitting based on a Gaussian distribution of formal potentials resulted in a standard deviation, sigma(E(o)’), of 40 mV for the cyt c/HOOC(CH2)(11)S/Au system. Physical models based on intrinsic surface heterogeneity and adsorption-induced heterogeneity arising from steric exclusion are proposed to explain the dispersion of formal potentials.