Cytochrome c was immobilized to several supports to study the effects of immobilization on the molecule and to serve as a model for the in vivo system. Immobilization was accomplished by covalent attachment of cytochrome c to the support surface, either Sepharose 6MB or glassy carbon. The effect of the coupling conditions on the covalent attachment reaction was studied with Sepharose 6MB. The reactive groups were monitored colorimetrically and were highly susceptible to hydrolysis. Correction for hydrolysis indicated that the covalent attachment reaction was first order with respect to reacted groups. Coupling conditions most affecting the amount of attached cytochrome c were the initial cytochrome c concentration, temperature, and pH. A detailed study of the resulting immobilized cytochrome c was conducted based on its three characteristic properties: spectra, oxidation reduction potential, and biological activity. The spectral properties demonstrated that no major conformational changes had occurred upon immobilization since the spectra were essentially the same. The redox potentials for most samples of immobilized cytochrome c loaded with different amounts of protein were found to be 20-25 mV lower than native cytochrome c (+ 270 mV). Two samples, the heaviest loaded, were approximately equal to the native protein suggesting that they may be least affected by immobilization. The biological activity measurements provide an indication of the ability of the molecule to function properly. The Michaelis constant (K(m)) for cytochrome oxidase and reductase with immobilized cytochromes c were significantly higher (20-400X) than the K(m) for soluble cytochrome c. The higher K(m)s reflect that about 1% of the immobilized cytochrome c is availble for reaction in agreement with distribution and exclusion studies. Correction of the immobilized cytochrome c K(m)s for available protein results in values similar to the soluble cytochrome c K(m). Immobilization of cytochrome c to glassy carbon was performed by two procedures employing a carbodiimide or 4-vinylpyridine as the coupling reagents. The former resulted in electrodes with higher specific activities and lower protein loadings than the latter. In both cases up to 60% of the immobilized protein was held by adsorption on the surface. Protein coverages were approximately 10⁻⁸ to 10⁻⁹ moles/cm² which corresponds to 100-800 layers.