Osteoblasts can be activated by their collagen matrix and in particular the DGEA peptide motif. We have reported that DGEA is able to activate Ca²⁺ signaling pathways in the human osteoblast-like cell line, Saos-2, by a tyrosine kinase-dependent pathway (T. J. McCann, W. T. Mason, M. C. Meikle, and F. McDonald. Matrix Biol. 16: 271-280, 1997). In the present study, we show that this activity is due to coupling of the signal to intracellular Ca²⁺ stores, since the DGEA action is not blocked by La³⁺ but is lost when Ca²⁺ stores are depleted with 2 µM and blocked by 10 µM ryanodine. The activated stores also differ functionally from those activated by thrombin, as blockade with U-73122 obstructs only thrombin-activated Ca²⁺ release. We have shown that the DGEA activity was not due to its high-charge density, since the two acidic residues can be substituted with their uncharged homologues (asparagine and glutamine) without significant loss of activity. This was in turn measured by an adhesion assay that also demonstrated this level of specificity. Furthermore, by constructing DGEA bound to FITC, we have shown that DGEA binding was dependent on divalent cations. We have also demonstrated that an intact actin cytoskeleton is not required for Ca²⁺ activation by inhibiting actin polymerization with the addition of cytochalasin B. These data strengthen the argument that collagen has a significant role in regulating osteoblast function via this peptide motif.