In this paper, we employ the surface-specific polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and sum-frequency generation (SFG) methods with surface pressure and surface potential isotherms to determine the organization of p-tert-butylcalix[6]arene molecules and their interaction with Cd(2+) ions in Langmuir monolayers. The area per molecule was estimated to be 135 Å(2), which corresponds to the Calix6 axis perpendicular to the air-water interface with most OH groups parallel to the interface. This area is larger than predicted by molecular modeling with quantum chemical calculations with a PM3 Hamiltonian (109 Å(2)), which is ascribed to the repulsion between Calix6 molecules. The incorporation of Cd(2+) ions in the subphase leads to drastic changes in the dipole moment contribution of the monolayer surface potential. Rather than increasing with incorporation of Cd(2+) ions owing to a decrease in the negative double-layer potential, the measured surface potential decreased monotonically with increasing ion concentration. This unexpected result was ascribed to a strong interaction with Cd(2+) ions that induced the calyx of the molecule to adopt a more open conformation at the air/water interface and affected the orientation of hydration water molecules, according to the SFG data. This finding allows us to understand the reason why the Gouy-Chapman model fails to explain surface potential results for subphases containing divalent or trivalent ions, and may be relevant for the application of calixarenes in sensing.