A comprehensive study of the bioavailability of orally administered silver nanoparticles (AgNPs) was carried out using a rat model. The silver uptake was monitored in liver and kidney tissues, as well as in urine and in feces. Significant accumulation of silver was found in both organs, the liver being the principal target of AgNPs. A significant (~50%) fraction of silver was found in feces whereas the fraction excreted via urine was negligible (<0.01%). Intact silver nanoparticles were found in feces by asymmetric flow field-flow fractionation (AsFlFFF) coupled with UV-Vis analysis. Laser ablation–ICP MS imaging showed that AgNPs were able to penetrate into the liver, in contrast to kidneys where they were retained in the cortex. Silver speciation analysis in cytosols from kidneys showed the metallothionein complex as the major species whereas in the liver the majority of silver was bound to high-molecular (70–25 kDa) proteins. These findings demonstrate the presence of Ag(I), released by the oxidation of AgNPs in the biological environment.