Basic helix-loop-helix (bHLH) transcription factors have been shown to play an important role in controlling cell type determination and differentiation. TWIST, a member of the bHLH transcription factor family, is involved in the development of mesodermally derived tissue, including the skeleton. We examined the role of human TWIST in osteoblast metabolism using stable expression of sense and antisense TWIST in human osteoblast HSaOS-2 cells. Changes in morphology and osteogenic phenotype characterized these stable clones. Cells that overexpressed TWIST exhibited a spindle shaped morphology, reduced levels of alkaline phosphatase, a reduced proliferation rate, and failed to respond to basic fibroblast growth factor (bFGF). In contrast, those that underexpressed TWIST demonstrated a cuboidal epithelial-like morphology characteristic of differentiated osteoblasts. TWIST antisense cells exhibited increased levels of alkaline phosphatase and type I collagen mRNA, initiated osteopontin mRNA expression, and had a reduced proliferation rate. These results indicate that TWIST overexpressing cells may de-differentiate and remain in an osteoprogenitor-like state, and antisense TWIST cells progress to a more differentiated mature osteoblast-like state. Therefore, the level of TWIST can influence osteogenic gene expression and may act as a master switch in initiating bone cell differentiation by regulating the osteogenic cell lineage.