The purpose of this study was to quantify differences in indentation modulus and microhardness between labeled osteons identified by epifluorescent microscopy and neighboring unlabeled osteons. In microradiographs and backscattered images, newly formed osteons appear more radiolucent (darker) than older osteons. This is ascribed to incomplete mineralization of the osteon. However, the mechanical properties of these young osteons are unknown. Nine femoral cross-sectional specimens were obtained from five skeletally mature dogs. Prior to death, the dogs received a pair of calcein bone labels. Labeled osteons were identified under an epiflourescent microscope. Bone specimens were transferred to a nanoindenter specimen holder, and the previous identified labeled osteons were located. Labeled (n = 102) and unlabeled (n = 101) osteons were examined by instrumented indentation testing. Indents were made to a depth of 500 nm at a loading rate of 10 nm/second. There were significant differences in the indentation modulus (P < 0.001) of labeled (10.02 +/- 3.61 gigapascal (GPa), mean +/- standard deviation) and unlabeled (15.11 +/- 3.72 GPa) osteons. Similar differences existed in microhardness measurements. Newly formed osteons had lower modulus (34%) and hardness (41%) than older osteons found in femoral cross sections. These data provide information on the indentation moduli of osteons during an early phase of mineralization compared to osteons that have completed mineralization.