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Collagen protein synthesis by osteoblasts is influenced by transforming growth factor-β (TGF-β1) and is essential to bone formation. The effectiveness of TGF-β1 depends on efficient delivery of the growth factor to target cells, adequate binding to cell surface receptors, and an optimum environment for promotion of collagen synthesis. The effects of calcium hydroxide (Ca(OH)2), TGF-β1, and Ca(OH)2/TGF-β1 co-administration on total protein, collagen protein, and noncollagen protein synthesis by early (subculture I) and late (subculture V) osteoblast cultures were tested. TGF-β1 significantly increased all protein synthesis in subculture I osteoblasts (p = 0.001; p < 0.001; p = 0.019). Ca(OH)2/TGF-β1 co-administration significantly increased total protein and collagen protein levels in subculture I osteoblasts as well (p = 0.048; p = 0.012). TGF-β1 increased total protein and collagen protein synthesis significantly in subculture V cells (p = 0.025; p = 0.01). These data indicate that co-administration of Ca(OH)2 and TGF-β1 enhances collagen synthesis by osteoblasts and may have implications for the clinical setting.