Real-time confocal particle tracking (CPT) was used to compare the transport and trafficking of polyethylenimine (PEI)/DNA nanocomplexes to that of efficient adenoviruses in live primary neurons. Surprisingly, the quantitative intracellular transport properties of PEI/DNA nonviral gene vectors are similar to that of adenoviral vectors. For example, the values of individual particle/virus transport rates and the distributions of particle/virus transport modes (i.e., the percentage undergoing active, diffusive, or subdiffusive transport) largely overlapped. In addition, both PEI/DNA vectors and adenoviruses rapidly accumulated near the cell nucleus in primary neurons despite our finding that PEI/DNA move slower in neurites than in the cell body, whereas adenoviruses move with equal rates in either location. The intracellular trafficking pathways of PEI/DNA and adenoviruses, however, were substantially different. The majority of PEI/DNA trafficked through the endolysosomal pathway so as to end up in late endosomes/lysosomes (LE/Lys), whereas adenoviruses efficiently escaped endosomes. This result suggests that the sequestration of nonviral gene vectors within acidic vesicles may be a critical barrier to gene delivery to primary neurons in the central nervous system (CNS).