Previous work in our group has shown that hydrophobic modification of chitosan and Nafion (R) membranes can be used to alter the transport properties of chemically modified electrodes. In this paper, we employ the same hydrophobically modified chitosan and Nafion (R) membranes to form and characterize enzyme modified electrodes for biofuel cell applications. This paper details the voltammetric characterization of the electrochemical flux at alcohol dehydrogenase, formate dehydrogenase, lactic dehydrogenase, glucose dehydrogenase, and formaldehyde dehydrogenase modified glassy carbon electrodes, where the enzymes were immobilized in both hydrophobically modified Nafion (R) and chitosan membranes where the degree of hydrophobic modification was systematically altered. The results conclude that the electrochemical flux is a function of both the transport and extraction properties of the membrane, as well as the size and catalytic activity of the enzyme. Finally, this paper details the first evidence that hydrophobically modified chitosan can be used at the anode of a biofuel cell. (C) 2007 Elsevier B.V. All rights reserved.