Numerous targeted nanotherapeutics have been described for potential treatment of solid tumors. Although attention has focused on antigen selection and molecular design of these systems, there has been comparatively little study of how cellular heterogeneity influences interaction of targeted nanoparticles with tumor cells. Antigens, such as HER2/ERBB2, are heterogeneously expressed across different indications, across patients, and within individual tumors. Furthermore, antigen expression in nontarget tissues necessitates optimization of the therapeutic window. Understanding the performance of a given nanoparticle under different regimens of antigen expression has the ability to inform patient selection and clinical development decisions. In this work, HER2-targeted liposomal doxorubicin was used as a model-targeted nanoparticle to quantitatively investigate the effect of HER2 expression levels on delivery of doxorubicin to the nucleus. We find quantitatively greater nuclear doxorubicin delivery with increasing HER2 expression, exhibiting a threshold effect at approximately 2 × 10(5) HER2 receptors/cell. Kinetic modeling indicated that the threshold effect arises from multiple low-affinity interactions between the targeted liposome and HER2. These results support previous data showing little or no uptake into human cardiomyocytes, which express levels of HER2 below the threshold. Finally, these results suggest that HER2-targeted liposomal doxorubicin may effectively target tumors that fall below traditional definitions of HER2-positive tumors, thereby expanding the potential population of patients that might benefit from this agent.