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The human pineal gland is the largest producer of the hormone melatonin. Pineal acervuli (brain sand), calcified concretions in the pineal gland, have long been studied because of their association with ageing, melatonin production, and neurological disorders. The solid inorganic matter of the hydroxyapatite crystals often renders sample sectioning impossible, to the extent that the sections lose value. Technically, freeze-fracturing has revealed the detailed structure and cell relationships without tissue damage. In our electron microscopic study, samples of the human pineal gland were obtained during autopsy from 20 donors with mean age 69 years. Samples underwent freeze-fracturing and standard histological procedures, and were analysed by scanning electron microscopy (SEM) in high vacuum. Based on our results, freeze-fracturing enabled identification of a mulberry-like acervulus topography. The acervuli were situated in specific “nest-like” structures, which were surrounded by pinealocytes, interstitial cells, and nerve fibres. A fractured surface of the intrapineal acervuli exhibited a regular lamellar structure. Freeze-fracturing the pineal gland and imaging by SEM enabled complex structural analysis. This approach permits viewing the surface acervuli spherical and internal lamellar architecture. Our results confirmed that the parenchyma of this small but important gland contains two types of acervuli, depending on their size: non-aggregated and aggregated. We propose to include these forms of acervuli in the new edition of the Terminologica Histologica. In conclusion, pineal gland freeze-fracturing by SEM is suitable for complex structural analysis. Our description of our methods can be a guide for other scientists who want to study the pineal gland with electron microscopy methods. Full article