The increasing uses of zinc oxide nanoparticles (ZnONPs) in coatings, paints, personal care products and many other products increase the possibility of the body's exposure to ZnONPs. Accurate and quantitative profiling on the tissue distribution and body clearance of ZnONPs, which is an important factor to clarify the acute and chronic safety concerns of ZnONPs, is interfered by the abundance of the body's endogenous zinc moiety. In this report, radioactive zinc oxide nanoparticles (R-ZnONPs) generated from neutron activation were employed for the in vivo bio-distribution studies using mice as the animal model. gamma-Ray emitting radioactive R-ZnONPs were produced from neutron activation. Zeta potentials of the ZnONPs before and after the neutron irradiation remained about the same, and R-ZnONPs largely remained its original nano-particulate form after neutron irradiation. After intravenous administration into ICR mice, R-ZnONPs exhibited a primary retention in lung (43.6% injected dose (ID)/g tissue wet weight) for the first hour and began to be translocated to intestinal tract for feces excretion at a later stage. This type of labeling free and radioactive nanoparticles retains the surface property and can be a convenient protocol for studying bio-distribution of nanoparticles in pristine chemical form.