p-tent-Butylcalix[4]arene-1,3-dithiol (BCAD) was synthesized to prepare a functionalized surface for host-guest molecular recognition by immobilizing it on the Au(100)-(1 x 1) surface in 0.05 M HClO4. In situ scanning tunneling microscopy (STM) was employed to investigate the structure of the adlayer of BCAD formed on the Au(100)-(1 x 1) surface. Highly ordered adlayers of BCAD with two different packing arrangements were found to form on the Au(100)-(1 x 1) surface. High-resolution STM images revealed the packing arrangement and even the internal molecular structure of BCAD.