Ribonucleic acid (RNA) homopolymer thin films on highly oriented pyrolytic graphite (HOPG) were prepared in ultrahigh vacuum (UHV) directly from aqueous solution by electrospray (ES) injection. The polyadenosine (poly rA) films were prepared in several steps of increasing thickness without breaking the vacuum. Before deposition and between deposition steps, the samples were characterized with photoemission spectroscopy (PES). Both X-ray and ultraviolet photoemission spectroscopy (XPS and UPS) were employed. XPS enabled the detailed measurement of core level peaks, giving insight into the chemical interaction at the interface and the layer morphology. The corresponding UP-spectra sequence allowed us to directly follow the transition from HOPG valence bands to the poly rA highest occupied molecular orbital (HOMO) structure. This enabled the determination of the poly rA ionization energy and work function as well as the charge injection barriers between the Fermi level of the HOPG substrate and the poly rA HOMO. The injection barrier between the lowest unoccupied molecular orbital (LUMO) and the HOPG Fermi level was determined using the HOMO-LUMO gap value determined by optical absorption. The results indicate that significant injection barriers exist between HOPG and the poly rA overlayer, limiting conductivity across this interface.