The dominant physical and geometrical parameters for imaging hydrated purple membranes with the scanning tunneling microscope are identified and characterized. These are sample voltage, current density, and tip radius. Purple membranes are imaged with vertical and lateral resolution of 0.7 nm and 15 nm, respectively. We also found that the apparent height of the membranes depends on the tip-substrate separation. These experiments pose the problem of electron transport through 5-10 nm thick insulating materials. We propose a model where the contrast mechanism is controlled by two factors, the electric field at the interface and the transmission through empty states in the membrane. © 1995, American Chemical Society. All rights reserved.