The purpose of this study was to evaluate a biodegradable hydroxyapatite/collagen composite and to examine the use of the calcium ion contained for bone formation and growth. Surgical holes were prepared in the femora and tibiae of beagle dogs, and were filled with the hydroxyapatite/collagen composite labeled with alizarin red. After 4 weeks, calcein was administered to the experimental dogs. After 1 additional week, the femora and tibiae were removed surgically and fixed in formalin. Light microscopy and confocal laser scanning microscopy were used to examine the surgical holes with their implanted materials and the surrounding bone. There were only a few inflammatory cells adjacent to the hydroxyapatite/collagen composite. The newly formed bone in the cortical bone was stained with calcein, which binds to serum calcium, and new bone near the hydroxyapatite/collagen composite in the holes was stained positive for alizarin red, which binds to the calcium in the hydroxyapatite/collagen composite. In addition, osteoblasts near the hydroxyapatite/collagen composite as well as newly formed bone adjacent to the osteoblasts showed alizarin red staining, but the new bone at a distance from the hydroxyapatite/collagen implant reacted only to calcein staining. These results, using the tissue labeling method with calcein and alizarin red, suggested that the calcium bound to the alizarin red released from the hydroxyapatite/collagen composite materials might have been translocated to sites of new bone formation. The present experiment showed that the novel hydroxyapatite/collagen composite is a useful implant material for bone augmentation and that the calcium in the newly formed bone might have been released from the implant.