Amphotericin B (AmB, 1) is a polyene antibiotic that has been used for treatment of systemic fungal infections. It is generally accepted that an ion-permeable channel formed across bilayer membranes is responsible for the pharmacological activity of AmB. A "barrel-stave" model has been most frequently discussed as the channel complex. Although many investigations about the channel structure have been carried out, details of the structures are not fully understood. To investigate the ion-channel structure, we applied solid-state NMR for evaluating the interaction between AmBs under membrane environments. First we prepared <13>^C-, <19>^F-, and 2^H-labeled AmBs [tri-^<13>C]AmB(4), derivatives 5 and 6, respectively) by biosynthetic or chemical synthetic methods. Then these labeled AmBs were mixed with DMPC and subjected to solid-state NMR measurements. The ^<13>C{<19>^F}REDOR spectra of DMPC membranes containing the ^<19>F-AmB and ^<13>C-AmB showed that the interatomic interactions were significantly weak in ergosterol-containing membrane as compared with those in sterol-free membrane. These results suggest that ergosterol enhances the mobility of AmB assemblies or changes intermolecular distances in AmB aggregates. To examine the mobility of AmB assemblies, solid-state 2^H-NMR was measured using 2^H-labeled AmB. The results showed ergosterol significantly increases mobility of AmB, while AmB is almost immobilized in sterol-free membrane. These results suggest that in sterol-free membranes AmBs are aggregated to undergo phase separation and in ergosterol-PC membranes AmBs form small assemblies, which may correspond to ion-permeable channels.