Gramicidin molecules were deposited on HOPG surfaces to characterize molecular orientation and film structure as a function of surface coverage and temperature. At low coverage (0.35 ML), the molecules adopted a flat-lying orientation and formed dendritic islands. At higher coverage (0.86 ML), molecules adopted an upright orientation and circular holes formed in the films. The upright film exhibited higher adhesion in force spectroscopy measurements, supporting our molecular orientation assignments. At elevated deposition temperatures (50 degrees C) on the higher coverage films, the holes were still present, but partially filled in. At 60 degrees C the film structure was quite different, forming tall irregular islands without the circular holes observed at lower temperatures. These results demonstrate that gramicidin molecular orientation and film structure on HOPG can be controlled by tuning the surface coverage and deposition temperature. (C) 2010 Elsevier Inc. All rights reserved.