To obtain atomic force microscopy (AFM) images of 5,11,17,23,29,35-hexa-tert-butyl-37,38,39,40,41, 42-hexakishydroxycalix[6]arene, a monolayer containing this macrocycle as well as octadecanol for monolayer stabilization was formed at the air/water interface by means of the Langmuir-Blodgett (LB) technique. The appropriate choice of the substrate onto which this monolayer was transferred for AFM observation was found to be crucial for high-resolution AFM. Whereas no images of the calix[6]arene deposited onto octadecyltrichlorosilane-treated mica could be obtained, FTS (1H,1H,2H,2H-perfluorodecyltrichlorosilane) treated mica was found to be an ideal substrate for the deposition and AFM observation. The large influence of the substrate on the resolution may be explained by the rigidness of the FTS layer as well as by the fact that the fee lattice of the chemisorbed FTS monolayer is commensurate with the arrangement of tert-butyl groups of the calix[6]arene. This method of sample preparation made possible the first AFM images of a macrocyclic host compound with molecular resolution and may prove to be of general applicability.