The enzyme catalase has been covalently bound to a self-assembled monolayer adsorbed to a gold substrate using a novel, organic solvent-based, attachment method. The enzyme was shown to remain catalytically active after immobilization to the surface. Under conditions of controlled humidity the effect of protein hydration on the scanning tunneling microscopy images of the naked molecules were investigated. Data were collected over a range of hydration conditions, from near zero to 86% relative humidity. Significant and reversible changes in the image quality were observed when the protein was dehydrated and hydrated relative to ambient humidity conditions. The findings are discussed in terms of the physical properties of hydrated proteins within the STM experiment.