The formation of inclusion complexes of cyclodextrins (CDs) and Methyl Orange (MO) has been examined by means of paper chromatography. On the basis of a proposed scheme in which there are an equilibrium between free MO and an inclusion complex in the mobile phase, an equilibrium between free MO in the mobile phase and that in the stationary phase of a chromatography paper, and an equilibrium between free inclusion complexes in the mobile and stationary phases, analyses have been made to evaluate these equilibrium constants. In the cases of α- and β-CDs, the equilibrium and the distribution constants for the formation of a 1:1 inclusion complex with MO are in good agreement with reported ones. The distribution of MO into the stationary phase of a chromatography paper is considerably prohibited by the formation of the inclusion complex. The estimated equilibrium constant for the formation of a 1:2 γ-CD-MO inclusion complex is an order of magnitude less than the reported one.