Vesicles composed of block copolymers (i.e., polymersomes) are one of the most versatile nano-carriers for medical purposes due to their tuneable physicochemical properties and the possibility to encapsulate simultaneously hydrophobic and hydrophilic substances, allowing, for instance, the combination of therapy and imaging. In cancer treatment, these vesicles need to remain long enough in the blood stream to be sufficiently taken up by tumors. Here, we have investigated the biodistribution and the pharmacokinetics of polymersomes, composed of poly(butadiene-b-ethylene oxide) having dimensions around 80 nm. The polymersomes have been radiolabeled with 111In via the so-called active loading method achieving a loading efficiency of 92.9 ± 0.9% with radionuclide retention in mouse serum of more than 95% at 24 h. The optimized 111In containing polymersomes have been intravenously administered in healthy and tumor bearing mice for pharmacokinetic determination using microSPECT (Single Photon Emission Computed Tomography). In healthy mice these polymersomes have been found to exhibit relatively long blood circulation (>6 h), low liver uptake (6 ± 1.5%ID/g, 48 h p.i.) and elevated spleen uptake (188 ± 30%ID/g). The blood circulation in tumor bearing mice is dramatically reduced (< 1.5 h) most likely due to elevated splenic filtration, clearly indicating the importance of in vivo studies in diseased mice. Finally, the polymersomes have been injected subcutaneously in tumor bearing mice revealing retention of 77% in the mice, primarily accumulated at the site of injection, up to 48 hours after administration.