Abstract In aqueous and aqueous d-glucose solutions, 6-anilino-2-naphthalenesulfonate (2,6-ANS) and 8-anilino-1-naphthalenesulfonate (1,8-ANS) form 1:1 inclusion complexes with β-CD. The molecular volume of 2,6-ANS bound to the β-CD cavity has been evaluated to be 4.7 nm3 from the viscosity dependence (Perrin plot) of the 2,6-ANS fluorescence polarization. This value indicates that 2,6-ANS incorporated into the β-CD cavity rotates as the inclusion complex on a fluorescence lifetime scale. The molecular volume of 2,6-ANS in solution without β-CD has been evaluated to be 0.88 nm3, which provides additional evidence for the whole rotation of the β-CD–2,6-ANS inclusion complex. The molecular volume of 1,8-ANS bound to the β-CD has been evaluated to be 2.7 nm3, which is comparable to the calculated molecular volume (1.9 nm3) of β-CD, indicating that 1,8-ANS most likely rotates as the inclusion complex. The fact that the molecular volume of the 1,8-ANS inclusion complex is smaller than that of the 2,6-ANS inclusion complex may be attributed to the differences in the binding site of β-CD and the substitution positions of anilino and sulfonato groups.