Mixed self-assembled monolayers (SAMs) composed of a medium length, reactive, n-organothiol (11-mercaptoundecanoic acid) and a short length, unreactive, hydrophobic n-alkylthiol (7-heptanethiol) chemisorbed onto evaporated gold surfaces, were used to study the effect of the microenvironment on the structure and activity of immobilized glucose oxidase (GOX). The mixed SAMs were characterized by X-ray photoelectron spectroscopy, contact angle measurements, and cyclic voltammetry. The derivatization of the SAMs for the covalent attachment and the immobilized enzyme were studied by X-ray photoelectron spectroscopy. Quantitative analysis of the amide I band of the infrared spectra of GOX immobilized onto surfaces at the two attainable extremes of the hyrdrophilicity range available to us indicated that the percentage of beta sheet increased with increasing hydrophilicity of the microenvironment. The specific activity of GOX was higher when the enzyme was immobilized onto the hydrophobic microenvironment.