Recently established techniques for performing in vivo micro-computed tomography (micro-CT) provide the capability of monitoring bone changes in a living animal at various points in time. However, radiation exposure from repeated micro-CT scans may have an effect on skeletal growth in normal or disease-model animals. The purpose of this study is to test a high resolution (approximately 10 microm) in vivo micro-CT protocol on mice and rats used for bone research to understand the impact of micro-CT radiation exposure on bone architecture. Ovariectomy (OVX) or sham-OVX surgery was performed on groups (n=6-8/group) of 12-week-old C3H/HeJ, C57BL/6J, and BALB/cByJ mice, and one strain of rat (Wistar, retired breeders). The right proximal tibiae were scanned at weekly intervals while the contralateral left limbs were not scanned until the endpoint of the protocol. Trabecular and cortical bone morphology was compared between radiated and non-radiated limbs at the endpoint to quantify the radiation effect. No effects of radiation were observed in OVX or sham rats. Lower trabecular bone volume was observed in the radiated limbs (-8 to -20% relative to non-radiated limb) of all mice groups except sham BALB/cByJ mice and normal control C57BL/6J mice, however, the observed effects were much less than the observed effects of ovariectomy ( approximately 40-50% total bone volume reduction, depending on mouse strain), and no interactions between radiation and OVX treatment were observed (p>0.2). Using an internal non-radiated control within each animal is a potential method to elucidate the effect of radiation exposure for any in vivo protocol. Thus, although in vivo micro-CT is a valuable tool for bone-related research, the impact of radiation in skeletally immature mice should be considered, particularly for strains with low bone volume at the measured site.