An experiment was designed to determine whether the material properties of fracture callus can be predicted by the measurement of mineral density. Rat tibiae that had a closed fracture were used to obtain uniform specimens of fracture callus at sequential stages of healing. An in vitro indentation test was done and tissue was biopsied, using a trephine, to determine the hardness and calcium content of a standardized area of callus tissue. The hardness of the callus was correlated with the mineral density of the local tissue, which initially had been measured non-invasively with single-photon-based micro-bone densitometry. The progress of differentiation and remodeling of callus was determined by comparative planimetric and microradiographic analyses of tissue. Determinations of density showed a linear correlation with concentrations of a hydroxyapatite equivalent solution (r = 0.997, p less than 0.001), with the ash weight of control specimens of cortical-trabecular bone (r = 0.998, p less than 0.001), and with the calcium content (micrograms per cubic millimeter) of the fracture callus (r = 0.854, p less than 0.001). The hardness of the callus had a non-linear relationship (r = 0.959, p less than 0.005) with the mineral density per volume of tissue, as measured by micro-bone densitometry. Clinical Relevance: The appearance of fracture callus on conventional radiographs is an important clinical finding in the assessment of fracture-healing. It is, however, an unreliable predictor of the strength of the union of the fracture. Three-dimensional imaging and quantification of bone mineral provide additional data about the quality and geometry of fracture callus. Our study showed that mineral content per unit of volume is a good index of hardness of the fracture callus. Thus, quantitative imaging may be useful clinically for monitoring fracture-healing.