Dodecyl ether derivatives 1-3 of p-sulfonatocalix[n]arene were incorporated into dimyristoyl phosphatidylcholine (DMPC) vesicles, and their binding abilities for acetylcholine (ACh) were examined by using steady-state fluorescence/fluorescence anisotropy and fluorescence correlation spectroscopy (FCS). For the detection of ACh binding to the DMPC vesicles containing 5 mol % of 1-3, competitive fluorophore displacement experiments were performed, where rhodamine 6G (Rh6G) was used as a fluorescent guest. The addition of Rh6G to the DMPC vesicles containing 3 resulted in a decrease in the fluorescence intensity of Rh6G with an increase of its fluorescence anisotropy, indicating that Rh6G binds to the DMPC- 3 vesicles. In the case of DMPC- 1 and DMPC- 2 vesicles, significant changes in the fluorescence spectra of Rh6G were not observed. When ACh was added to the DMPC- 3 vesicles in the presence of Rh6G ([ 3]/[Rh6G]=100), the fluorescence intensity of Rh6G increased with a decrease in its fluorescence anisotropy. From the analysis of fluorescence titration data, the association constants were determined to be 7.1×10 5 M -1 for Rh6G- 3 complex and 1.1×10 2 M -1 for ACh- 3 complex at the DMPC- 3 vesicles. To get a direct evidence for the binding of Rh6G and its displacement by ACh at the DMPC- 3 vesicles, diffusion times of the Rh6G were measured by using FCS. Binding selectivity of the DMPC- 3 vesicles for ACh, choline, GABA, l-aspartic acid, l-glutamic acid, l-arginine, l-lysine, l-histamine and ammonium chloride was also evaluated using FCS.