We previously reported that materials leached from bioactive glass (BG) and vitamin D, induced the formation of nodules on cultured periodontal-ligament fibroblasts (PLF). In this study, we have investigated the relationship between the conditions of the materials and nodule formation, analyzed morphologically, and also studied whether the production of nodules was specific to cultured PLF. PLF and skin fibroblasts were cultured in the presence or absence of BG. The amounts of calcium, phosphate, sodium and silicon in the culture medium and the number of nodules were measured at the 55th day. The nodules were observed microscopically and analyzed using an X-ray mi-croanalyzer. In PLF, nodules were formed regardless of the presence or absence of BG; however, they were more numerous in the presence of BG. In skin fibroblasts, nodules were not observed. The amounts of calcium and silicon were higher in the presence of BG, while the amount of phosphate was lower. The nodules appeared crystalline with a spongy structure and contained calcium and phosphorus. Our results show that the nodules were associated with PLF and precipitated by the materials (higher concentrations of calcium and silicon), and they were spongy crystal composed of calcium and phosphorus.