The ultrastructure of the secretory endpiece of the rat sublingual gland was examined in samples prepared by rapid freezing and freeze-substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphics from light micrographs of serial sections. Fixation by rapid freezing followed by freeze-substitution preserved cellular ultrastructures, especially the membrane structure, in perfect condition, and demonstrated the terminal portion of the sublingual gland to be a compound branched tubulo-alveolar gland with serous cells distributed throughout the end-pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure. In contrast, samples fixed by the conventional immersion method showed distended mucous cells displacing the serous cells toward the basal portion of the acinus to form the demilune structure. The luminal space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous cells during immersion fixation.