Selective molecular interactions at an interface formed by self-assembly of a macrocyclic synthetic host, calix[4]resorcinarene with four thiol groups (tetrathiol-CALM), are investigated. The recognition of guest adsorbates from aqueous solutions is monitored using surface plasmon resonance (SPR), and the orientation of the guest molecule is probed using polarization modulation infrared reflection absorption spectroscopy. Association of a series of lactones reveals that SAMs of the tetrathiol-CALM can discriminate between guest molecules with small differences in pendant molecular groups on the five-membered cyclic ring. SPR results show that the rate of association on the tetrathiol-CALIX surface is slower than that reported in the literature for a similar host where the monolayer is more ordered and the host cavity is made rigid using an O-bridged aromatic cavity. The differences indicate that the order in the monolayer and the rigidity of the cavity are important aspects in the recognition process. Strong and irreversible association of a guest molecule containing hydroxyl moieties points to the importance of hydrogen bonding effects. The absence of association with a hydrophilic interface formed from the self-assembly of mercaptoundecanol shows that the guest-host recognition is also driven by factors such as hydrophobic interactions and steric match. These factors permit selective discrimination between two structural isomers by the self-assembled surfaces of the synthetic host.