Nanoparticles for medical applications are frequently administered via parenteral administration. In this study, the tissue distribution of three lipid formulations based on Nanostructured Lipid Carriers (NLCs) after intravenous administration to rats was evaluated. NLCs were prepared by a high pressure homogenization method and varied in terms of particle size, surface charge, and surfactant content. The 99mTc radiolabeled NLCs were intravenously administered to rats, and radioactivity levels in blood and tissues were measured. Cmax, AUC0–24, and MRT0–24 were obtained from the radioactivity level versus time profiles. The radiolabeled nanocarriers exhibited a long circulation time since radioactivity was detected in blood even 24h post-injection. No differences on the MRT values in blood among the NLCs were observed, in spite of the different particle size and surface charge. The highest radioactivity levels were measured in the kidney, followed by the bone marrow, the liver, and the spleen. In the kidney, there was a higher accumulation of the positive nanoparticles, and in the liver, uptake of negative nanoparticles was higher than positive ones. NLCs with the largest particle size showed a higher uptake in the lung and lower accumulation in liver and bone marrow, in comparison with the smaller ones.