Two distinct IR spectra of amphotericin B have been reported. These differences can be obtained from the same sample by surprisingly small changes in the method of sample preparation. Type I spectra (hand-ground samples) are characterized by a sharp CO stretching band at 1692 cm−1, and Type II spectra (vibrator-ground samples) are characterized by a broad CO stretching band near 1710 cm−1. X-ray powder diffraction demonstrates that vibrator grinding promotes a transition from a crystalline to an amorphous phase. The two phases are not bioequivalent. Differential thermal analysis reveals a transition near 157°, and samples heated to 158° give only Type II IR spectra. However, a marked color change accompanies such heating (i.e., structural changes affecting the chromophore have been thermally induced), while X-ray spectra show an increase of only about 30% in amorphous content. Furthermore, hand-ground samples heated to 120° still display only Type I IR spectra. Thus, the vibrator-induced transition is not solely a static thermal effect. Many observed spectral lines can be assigned to specific functional groups.