Adhesion-induced changes in intracellular calcium concentration ([Ca2+]i) were measured in populations of human osteoblasts spreading on bone matrix proteins. In cells spreading on collagen type I, fibronectin, or laminin, average values for [Ca2+]i were found to increase approximately 2x over baseline and then decline. The speed with which [Ca2+]i increased and declined was dependent upon the matrix protein on which the cells were plated but was generally complete within 1 hour from the time of plating. Calcium mobilization was found to be due to influx of calcium across the osteoblast plasma membrane and was integrin dependent. Carboxyamido triazole (CAI), a specific inhibitor of nonvoltage-dependent calcium channels, or BAPTA-AM, a chelator of intracellular calcium, inhibited osteoblast adhesion and spreading on collagen type I, fibronectin and laminin in a dose-dependent manner. In conclusion, these results demonstrate that calcium mobilization is induced upon integrin-ligand contact and that calcium influx is required for cell adhesion and spreading.