Background : Features of the tumor microenvironment influence the efficacy of cancer nanotherapeutics. The ability to directly radiolabel nanotherapeutics offers a valuable translational tool to obtain biodistribution and tumor deposition data, testing the hypothesis that the extent of delivery predicts therapeutic outcome. In support of a first in-human clinical trial with 64Cu-labeled HER2-targeted liposomal doxorubicin (64Cu-MM-302), a preclinical dosimetric analysis was performed. ; Methods : Whole-body biodistribution and pharmacokinetic data were obtained in mice that received 64Cu-MM-302 and used to estimate absorbed radiation doses in normal human organs. PET/CT imaging revealed non-uniform distribution of 64Cu signal in mouse kidneys. Kidney micro-dosimetry analysis was performed in mice and squirrel monkeys, using a physiologically based pharmacokinetic model to estimate the full dynamics of the 64Cu signal in monkeys. ; Results : Organ-level dosimetric analysis of mice receiving 64Cu-MM-302 indicated that the heart was the organ receiving the highest radiation absorbed dose, due to extended liposomal circulation. However, PET/CT imaging indicated that 64Cu-MM-302 administration resulted in heterogeneous exposure in the kidney, with a focus of 64Cu activity in the renal pelvis. This result was reproduced in primates. Kidney micro-dosimetry analysis illustrated that the renal pelvis was the maximum exposed tissue in mice and squirrel monkeys, due to the highly concentrated signal within the small renal pelvis surface area. ; Conclusions : This study was used to select a starting clinical radiation dose of 64Cu-MM-302 for PET/CT in patients with advanced HER2-positive breast cancer. Organ-level dosimetry and kidney micro-dosimetry results predicted that a radiation dose of 400 MBq of 64Cu-MM-302 should be acceptable in patients.