Mixed per-6-thio-beta-cyclodextrin (CD-SH)/pentanethiol (C5SH) monolayers were constructed by the sequential immersion of a Au(111) electrode into solutions of CD-SH, ferrocene, and a mixed solution of ferrocene and C5SH. Highly compact CD-SH self-assembled monolayers (SAMs) with the surface CD-SH density of 74.0 +/- 6.3 pmol cm(-2) on a true surface area basis were formed in 1 mM CD-SH with the immersion time of more than 48 h as confirmed by reductive desorption voltammetry. Based on the concentration dependence of the adsorption amount, a Langmuir adsorption coefficient was determined to be 1.9 x 10(7) M-1. Chronocoulometry in a ferrocene solution at the CD-SH SAM and the mixed CD-SH/C5SH monolayers revealed the following inclusion properties. (1) All the CD-SH cavities can be used for the inclusion of a guest compound before and after the adsorption of C5SH, as shown by the fact that the maximum inclusion amounts of ferrocene, 68.0 +/- 3.4 and 73.0 +/- 2.0 pmol cm(-2) before and after the adsorption of C5SH, respectively, were very close to the surface CD-SH density, (2) The association constant between the surface-con fined CD-SH and ferrocene (7.6 x 10(4) M-1) is greater than the corresponding association constant in solution. (3) The intermolecular vacancies between the adsorbed CD-SH molecules are completely filled with C5SH. This ensures that the CD cavities are the only accessible sites for guest compounds and any other reactants.