In bone fracture healing, the extent to which the injured bone regains stability and strength depends on the mechanical properties of the tissues that are formed during healing. While many techniques have been used to quantify the overall mechanical behavior of fracture calluses, few data exist on the material properties of individual callus tissues. The overall goal of this study was to quantify these material properties. Nanoindentation was performed at multiple locations across thin (200mum), longitudinal sections of rat fracture callus at 35 days post fracture. Following indentation, sections were stained with alizarin red S and alcian blue to obtain semi-quantitative estimates of tissue mineral content and proteoglycan content, respectively. Indentation moduli varied over three orders of magnitude (0.61-1010MPa) throughout the callus. Much of this variation was due to the presence of multiple tissue types: the indentation moduli of granulation tissue, chondroid tissue and woven bone ranged 0.61-1.27MPa (median=0.99MPa), 1.39-4.42MPa (median=2.89MPa) and 26.92-1010.00MPa (median=132.00MPa), respectively. In regions of alizarin red staining, the indentation modulus was correlated (r=0.62, P=0.04) with stain intensity, suggesting a positive correlation between modulus and mineral content in woven bone. In addition, the indentation modulus of woven bone along the periosteal aspect of the cortex increased with distance from the fracture gap (P=0.004). These results demonstrate the usefulness of nanoindentation in characterizing the elastic properties of the heterogeneous mixture of tissues present in bone fracture callus.