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In this paper, changes of microstructural characteristics of disperse systems during mechanical activation of zinc oxide (ZnO) have been investigated. ZnO powder was activated by grinding in a planetary ball mill in a continuous regime in air during 300 min at the basic disc rotation speed of 320 rpm and rotation speed of bowls of 400 rpm but with various balls-to-powder mass ratios. During ball milling in a planetary ball mill, initial ZnO powder suffered high-energy impacts. These impacts are very strong, and large amounts of microstructural and structural defects were introduced in the milled powders. The morphology and dispersivity of particles and agglomerates of all powders were investigated by scanning electron microscopy and scanning transmission electron microscopy. The specific surface area of initial ZnO powder was determined as 3.60 m2 g−1 and it increased to 4.42 m2 g−1 in mechanically activated powders. An increase of the ball-to-powder mass ratio led to a decrease of particle dimensions as well as increased the tendency for joining into quite compact agglomerates, that is aggregates, compared with the very loose, soft initial agglomerates. The obtained results pointed out that activation of ZnO powders produces a highly disperse, nano-scaled mixture of small particles, that is crystallites with sizes in the range of 10–40 nm. Most of these particles are in the form of aggregates with dimensions of 0.3–0.1 μm. The crystallite and aggregate size strongly depend on milling conditions, that is ball-to-powder mass ratio, as shown in this investigation.