The effects of a high protein diet on bone formation and calcium (Ca) metabolism were evaluated in rats using an ectopic endochondral bone induction model. A control diet (18% casein) or a high protein diet (18% casein + 20% lactalbumin) was given to 50-day-old rats. Ten days after the feeding of the experimental diet, rats were intramuscularly implanted with demineralized bone powder (day 0). On day 14 and day 21, the implanted bone powder was harvested, and blood and urine samples were also obtained. Urinary Ca excretion was not increased on day 12-14; however, it was elevated on day 19-21 in rats fed the high protein diet compared with rats fed the control diet. The high protein diet remarkably stimulated urinary sulfate excretion in both sampling periods, which reflected dietary sulfur-containing amino acids contents. Also, rats fed the high protein diet exhibited a decrease in serum Ca concentrations. There was little difference in Ca contents and the activities of alkaline phosphatase and acid phosphatase in the implants between control group and high protein diet group on day 14 and day 21. Histological examination in the implanted demineralized bone powder on day 14 indicated only cartilage in rats fed the high protein diet in contrast to the occurrences of osteogenesis and remodeling in those fed the control diet. Retarded bone formation in rats fed the high protein diet might be owing to, in part at least, a restricted amount of Ca utilized at the stage of cartilage calcification.