PEGylated nanoparticles and macromolecules are increasingly used in cancer imaging and anticancer treatment. The role of receptor-mediated endocytosis in the efficacy of these agents, however, has not been clearly defined. Here, we developed a matched pair of endocytic and nonendocytic receptors to directly and unambiguously assess this issue. The ligand-binding domains of the low-density lipoprotein receptor (LDLR) or a truncated LDLR lacking the NPXY endocytosis motif (DeltaLDLR) were replaced with an anti-polyethylene glycol antibody (alphaPEG) to form endocytic alphaPEG-LDLR and nonendocytic alphaPEG-DeltaLDLR receptors. The receptors were stably expressed at similar levels on the surface of HCC36 cells. HCC36/alphaPEG-LDLR cells, but not HCC36/alphaPEG-DeltaLDLR cells, rapidly endocytosed PEG-quantum dots and PEG-liposomal doxorubicin (Lipo-Dox) in vitro and in vivo. Lipo-Dox was significantly more cytotoxic to HCC36/alphaPEG-LDLR cells than to HCC36/alphaPEG-DeltaLDLR cells. HCC36/alphaPEG-LDLR tumors also accumulated significantly more PEGylated near-IR probes (PEG-NIR797) and PEG-liposomal-(111)In than HCC36/alphaPEG-DeltaLDLR tumors in vivo. Furthermore, Lipo-Dox more significantly suppressed the growth of established HCC36/alphaPEG-LDLR tumors as compared with HCC36/alphaPEG-DeltaLDLR tumors. Our data show that endocytosis of PEGylated probes and drugs enhances both cancer imaging and anticancer efficacy, indicating that endocytic receptors are superior targets for the design of cancer imaging probes and immunoliposomal drugs.