Stable integration of collagenous tissue-engineered constructs to surrounding solid devices can be accomplished by coating the solid surfaces with exogenous alkaline phosphatase (ALP). We showed previously that coating of culture well surfaces with the enzyme in combination with the presence of its substrate β-glycerophosphate (β-GP) induces mineral deposition at the interface of matrix and surface, thereby preventing matrix detachment. In this study the effect of such mineral-inducing conditions on differentiation of human periodontal ligament (PDL) fibroblasts into osteoblasts/cementoblasts was analyzed in three-dimensional collagen gels. Mineral-inducing conditions decreased collagen type I gene expression and induced dentin matrix protein 1 (DMP1; a marker of late osteoblasts/cementoblasts) gene expression by fibroblasts. DMP1 protein was detected in some fibroblasts only in mineralizing gels. Exogenous ALP released high levels of inorganic phosphate from β-GP. Addition of inorganic phosphate alone induced DMP1 gene expression, which could be prevented by blocking phosphate entry into fibroblasts by foscarnet. We concluded that mineralizing conditions induced by exogenous ALP affect the phenotype of PDL fibroblasts. The fibroblasts are stimulated to express the late osteoblast/osteocyte marker protein DMP1, which is mediated by uptake of inorganic phosphate into the cells. The enzyme-mediated mineral deposition may thus facilitate enhanced integration of collagenous tissue-engineered constructs to devices or implants in vitro.