MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of beta-glycerophosphate (beta-GP). In the present work, we study the influence of concentration and duration of beta-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following beta-GP pulse. With continuous treatment at 5 mmol beta-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower beta-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with beta-GP. These data indicate that, in viable cells, high doses of beta-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.