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Gold nanoparticles (GNPs) are biocompatible nanomaterials that are currently researched for biomedical applications such as imaging and targeted drug delivery. In this investigation, we studied the effects of a single dose (injected on day 1) as well as a priming dose (two injections with a gap of one week) of 5 nm, 20 nm, and 50 nm diameter GNPs on the structural and biochemical changes in the liver, kidney, and spleen of mice. The results showed that small sized GNPs (5 nm) produced significant pathological changes in the liver on day 2 that gradually reduced on day 8. The medium (20 nm) and large (50 nm) sized GNPs preferentially targeted the spleen and caused significant pathological changes to the spleen architecture on day 2 that persisted on day 8 as well. There were minimal and insignificant pathological changes to the kidneys irrespective of the GNPs size. The animals that were primed with the pre-exposure of GNPs did not show any aggravation of histological changes after the second dose of the same GNPs. None of the dose regimens of the GNPs were able to significantly affect the markers of oxidative stress including glutathione (GSH) and malondialdehyde (MDA) in all of the organs that were studied. In conclusion, the size of GNPs plays an important role in their pathological effects on different organs of mice. Moreover, the primed animals become refractory to further pathological changes after the second dose of GNPs, suggesting the importance of a priming dose in medical applications of GNPs. [ABSTRACT FROM AUTHOR]