Nanotechnology has been used to provide advanced biomedical research tools in diagnostic imaging and therapy, which requires targeting of nanoparticles (NPs) to individual cells and subcellular compartments. However, a complete understanding of the intracellular uptake, transport, and subcellular distribution of nanostructured materials remains limited. Hence, gold NPs were explored as a model system to study the intracellular behavior of NPs in real time. Our results show that the cellular uptake of gold NPs is dependent on their size and surface properties. The NPs were transported in vesicles of 300-500 nm diameter within the cytoplasm. The average velocity and diffusion coefficient of the vesicles containing NPs were 10.2 (+/-1.8) microm/hr and 3.33 (+/-0.52) microm 2/hr, respectively. Analysis of the time-dependent intracellular spatial distribution of the NPs demonstrated that they reside in lysosomes (final degrading organelles) within 40 minutes of incubation. These findings can be used to tailor nanoscale devices for effective cell targeting and delivery.