Tenascin-W is a glycoprotein secreted into the extracellular matrix of developing bones. Here, we have examined possible roles for tenascin-W in osteogenesis. Purified recombinant tenascin-W, like tenascin-C, increases the number of mineralized foci in primary cultures of avian osteoblasts and increases alkaline phosphatase activity in vitro. In addition, tenascin-W in solution promotes the migration of primary osteoblasts across fibronectin-coated filters. The sixth fibronectin type III domain of chicken tenascin-W contains a phylogenetically conserved KGD motif that is predicted to be available to integrin binding. To determine whether this motif is potentially functional, we have cultured osteoblasts on KGD-containing peptides and control peptides. Osteoblasts cultured on peptides with the KGD motif acquire a multipolar phenotype with pseudopods tipped with actin-rich ruffles, which is similar to the morphology of osteoblasts cultured on recombinant tenascin-W. Moreover, the KGD peptides, but not the control peptides, promote proliferation in cultured osteoblasts but not alkaline phosphatase activity or migration. Finally, explanted embryonic frontal bones are significantly thicker when cultured in the presence of tenascin-W, suggesting that tenascin-W can accelerate the formation of new bone in a complex multicellular environment.