Osteoblasts derived from Day 21 fetal rat calvaria grown on films of collagen type I exhibit an earlier and enhanced expression of the differentiated phenotype, compared to cells cultured on plastic. The temporal expression of genes characterizing three distinct periods of growth and differentiation are dramatically modified. During the initial proliferation period, expression of genes normally expressed at high levels on plastic (fibronectin, beta 1 integrin, and actin) was decreased from 50 to 70% in cells grown on collagen. Genes normally expressed at maximal levels in the postproliferative period (osteonectin, osteocalcin, and osteopontin) were up-regulated severalfold very early. Alkaline phosphatase enzyme activity was elevated 2- to 3-fold during the proliferation period, while mRNA levels remained low, suggesting post-transcriptional modifications. The most dramatic consequence of culture of cells on collagen is the accelerated and uniform mineralization of the matrix in contrast to the focal mineralization confined to bone nodules in cultures on plastic. Type I collagen supports maintenance of osteoblast phenotypic properties of passaged cells in the absence of glucocorticoid supplementation required for differentiation of osteoblasts subcultivated on plastic. Treatment of proliferating rat osteoblasts on plastic with 1,25(OH)2D3 blocks osteoblast differentiation and matrix mineralization. Although differentiation-related genes (alkaline phosphatase and osteocalcin) were up-regulated by vitamin D, culture on the collagen matrix could not overcome the inhibition of mineralization. Taken together, these studies define the critical role of type I collagen in mediating the signaling cascade for expression of a mature osteoblast phenotype and mineralization of the extracellular matrix in a physiological manner.