Human dental papilla cells were enzymatically separated from deciduous tooth germs of an 8-month-old embryo legally aborted. The second passage cells were cultured up to 35 days in 3 groups. The beta-GP group was cultured in the Dulbecco MEM containing ascorbic acid and beta-glycerophosphate supplemented with 15% fetal bovine serum. The Dex group was in the same medium, in addition containing dexamethasone. The control group contained none of the 3 chemicals. Mineralized nodules were formed after 15 days in the beta-GP and Dex groups. Only in the presence of ascorbic acid and organic phosphate did they mineralize. The addition of dexamethasone caused a significant increase in the number of nodules. By electron microscopy, the nodules contained needle-shaped crystals associated with a network of collagen fibrils. Calcium and phosphorus were detected by energy-dispersive X-ray microanalysis in the nodules. Furthermore, the crystalline material exhibited a pattern consistent with hydroxyapatite and dentin when examined by X-ray diffractometry. Cells showed high levels of alkaline phosphatase activity, which was increased 2-3 times in the presence of the 3 chemicals. These results indicated that human dental papilla cells have the ability to form dentin in culture. The formation of mineralized nodules by human dental papilla in vitro provides a useful model for studying the morphogenesis and differentiation of dental papilla ectomesenchyme.