As part of ongoing studies aimed at clarifying the early events of bone matrix deposition and mineralization, we have characterized primary osteoblast cultures using ultrastructural and immunocytochemical methods. Osteogenic cells were isolated by sequential enzymatic digestion of newborn rat (2-4-day-old) calvariae and grown for periods of 7 to 28 days on polystyrene, Thermanox plastic, or sputtered titanium. Bone-like nodules, showing a stratified organization of cells and collagen, were examined by scanning and transmission electron microscopy, and further characterized for mineral by backscattered electron imaging and X-ray microanalysis. Colloidal gold immunocytochemistry was used to examine the distribution of osteopontin in these nodules. Cells at the surface of the nodules were rounded, while those within the nodules generally appeared more flattened. Both cell types, particularly at early culture intervals, exhibited well-developed protein synthetic organelles. Collagen fibrils were present between the cell layers and some individual fibrils appeared mineralized. Aggregates of needle-shaped crystallites were sometimes apposed to the cell surface, frequently within invaginated regions of the cell membrane, while other mineralized masses of various sizes were present within the collagenous scaffolding. The periphery of the mineralized masses was often delimited by an electron-dense, lamina Limitans-like layer. Focal accumulations and/or a more complete layer of afibrillar, mineralized organic matrix were sometimes observed at the interface between the cells and the surface of the culture dish. Osteopontin was immunodetected over the afibrillar and collagenous mineralized matrix throughout the cultures and, in some cases, labeling was concentrated over the peripheral, electron-dense material delimiting the mineralized masses. In conclusion, these data indicate that calvaria-derived osteoblasts produce an extracellular matrix with structural and compositional similarities to bone. Although not a regular observation, the accumulation of osteopontin on the surface of the culture substrate and at the periphery of masses of mineralized matrix may be analogous to what takes place in vivo at naturally occurring bone interfaces.