Untreated specimens (i.e. not fixed, dehydrated or embedded) of the otolithic membrane from the sacculus of guinea pigs were observed at the ultrastructural level by low-vacuum scanning electron microscopy (LVSEM). This technique revealed the presence of a 15- to 20-µm-thick layer of an amorphous substance (the supraotolithic cupula zone) on the surface of the otoliths, which was not detectable by conventional methods. Elemental analysis of this substance revealed relatively high concentrations of oxygen, sodium, phosphorus, chlorine, potassium and calcium. This amorphous substance was thought to have a role in fixing the otoliths onto the sensory epithelium. In addition, the tips of the triangular portions of the otoliths were not sharp as shown by conventional SEM and were seen to be more rounded by LVSEM