Compartmentalization within eukaryotic cells hinders the efficient delivery of therapeutic agents to the cell nucleus. Here we describe novel multifunctional DNA carriers (MDCs) that self-assemble with DNA to form structured nanoparticles that possess virus-like functions for cellular trafficking. MDCs contain, in fusion, the DNA-compacting sperm chromatin component protamine, alpha-stimulating hormone for cell-targeted internalization, the endosome-translocation domain of diphtheria toxin, and an optimized nuclear localization sequence to confer recognition by the nuclear import machinery. The structure of the MDC-DNA particles was examined using atomic force microscopy, and the functionality of each domain assessed using in vitro techniques, including a reconstituted nuclear transport assay in semi-intact cells relying on the use of quantitative confocal laser scanning microscopy. The nanoparticles were internalized in cell-specific fashion and subsequently exited the endosome into the cytoplasm. Notably, the nanoparticles interact with cellular nuclear transport proteins as shown in direct binding assays and are actively trafficked into the cell nucleus of nondividing cells, resulting in 3- to 4-fold higher reporter gene expression in growth-arrested human embryonic kidney cells, as well as lower cytotoxicity, than lipid and polyethyleneimine vectors. The importance of each functional domain was examined by comparing MDCs with different domain compositions as controls, as well as using antibodies to block particular pathways. MDCs that utilize cellular signaling pathways have enormous potential to safely and efficiently deliver therapeutic transgenes into the nucleus of nondividing cells.-Glover, D. J., Ng, S. M., Mechler, A., Martin, L. L., Jans, D. A. Multifunctional protein nanocarriers for targeted nuclear gene delivery in nondividing cells.