This study examines the capability of NIH3T3 fibroblasts to express osteoblastic markers following stimulation with a number of hormones and growth factors in vitro. Of the agents tested, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently induced alkaline phosphatase (ALP) activity in NIH3T3 cells, and this effect was enhanced by the addition of dexamethasone (Dex), which when administered alone caused no detectable ALP expression. The combined use of 1,25(OH)2D3 and Dex also stimulated the synthesis of osteocalcin, and osteopontin. Furthermore, cells treated with the both hormones, in the presence of β-glycerophosphate and l-ascorbic acid, formed mineralized plaques, indicating an osteoblast (OB) phenotype. By contrast, the differentiation induced by 1,25(OH)2D3 or 1,25(OH)2D3 plus Dex was significantly antagonized by transforming growth factor-β1 and all trans-retinoic acid. These data indicate that NIH3T3 cells have the potential to adopt an OB-like phenotype and may prove to be a convenient model for studying the early events of osteogenic differentiation and the specific interactions of 1,25(OH)2D3 with glucocorticoids in controlling this process in vitro. © 2002 Wiley-Liss, Inc.