In rats with induced rickets, the uptake of 99mTcO4 and 99mTc-pyrophosphate per gram of bone was increased as compared with weight-matched controls. However, the uptake of radioactive calcium and 32P-pyrophosphate was similar in both rachitic and control animals, suggesting that the 99mTc label conferred specificity and favored the rachitic lesions. Employing the rat tibia in an in vitro system, 99mTcO4 uptake was predominantly in the organic bone matrix; radioactive calcium, 32P-pyrophosphate, or 14C-diphosphonate uptake was mainly in the bone mineral; and 99mTc-pyrophosphate, 99mTc-diphosphonate, and 99mTc-polyphosphate were found in both mineral and organic phases. By removal of both mineral and polysaccharide and by using agents that altered the degree of collagen fibril cross-linking, evidence was obtained suggesting the 99mTcO4 and 99mTc-pyrophosphate are preferentially bound by immature collagen.