To evaluate the sequential ultrastructural pathogenesis of the increase in osseous tissue and hyperosteoidosis previously demonstrated in rats administered supraphysiologic doses of 1,25(OH) 2D 3 and fed high levels of dietary calcium, young adult female rats were placed on a 2.5% calcium and 0.3% phosphorus diet, administered ethanol or 135 ng (5 units) 1,25(OH) 2D 3 IP daily, and killed after 2,4,6,8, and 10 days. Metaphyseal trabeculae from 1,25(OH) 2D 3 and placebo-treated rats were examined. Osteoblast hypertrophy characterized by increased cytoplasmic area, abundant rough endoplasmic reticulum, and prominent Golgi apparatus was evident in 1,25(OH) 2D 3-treated rats at Day 4. These osteoblasts were interpreted to be active in matrix synthesis. Widened osteoid seams were present at Day 6. Osteoblast hypertrophy and widened osteoid seams persisted through Day 10 in 1,25(OH) 2D 3-treated rats. The unmineralized bone matrix in 1,25(OH) 2D 3-treated rats contained more numerous cytoplasmic processes from adjacent osteoblasts than did control animals and loosely arranged collagen fibrils, which failed to aggregate in regions adjacent to the osteoid-mineralized bone interface as in placebo-treated rats. Osteoid seams in 1,25(OH) 2D 3-treated rats contained irregular electron-dense foci, which were often concentrated around embedded cytoplasmic processes. Osteocytic hypertrophy characterized by increased cytoplasmic area, developed rough endoplasmic reticulum, and increased numbers of mitochondria was evident at Day 2 and was sustained through Day 10 in 1,25(OH) 2D 3-treated rats. Variable-sized aggregates of electron-dense deposits similar to those concentrated around osteoblast cytoplasmic processes were observed in the pericellular space and on and immediately adjacent to the plasma membranes of osteocytes and embedding osteoblasts in 1,25(OH) 2D 3-treated rats as early as Day 4. The composition of these deposits is uncertain, but their electron density was only partially reduced by decalcification and omission of osmium postfixation. Semiquantitative energy-dispersive x-ray microanalytic studies demonstrated the absence of calcium in deposits located on or immediately adjacent to the plasma membranes, with minimal amounts of calcium being detected in deposits more removed from the plasma membranes. Osteoclasts were infrequent at Days 8 and 10 in 1,25(OH) 2D 3-treated rats compared with placebo-treated rats. These ultrastructural findings support previous quantitative light microscopic studies that show that the increase in osseous tissue and hyperosteoidosis in rats fed a high-calcium diet and administered supraphysiologic doses of 1,25(OH) 2D 3 is due in part to a decrease in osteoclasts and enhanced osteoblastic synthetic activity. Osteoblast hypertrophy in association with widened osteoid seams suggests increased bone formation. The bone matrix synthesis induced by 1,25(OH) 2D 3 and its subsequent mineralization appear to be abnormal.