Nanofluidic architectures and devices have already had a major impact on forefront problems in chemical analysis, especially those involving mass-limited samples. This critical review begins with a discussion of the fundamental flow physics that distinguishes nanoscale structures from their larger microscale analogs, especially the concentration polarization that develops at nanofluidic/microfluidic interfaces. Chemical manipulations in nanopores include nanopore-mediated separations, microsensors, especially resistive-pulse sensing of biomacromolecules, fluidic circuit analogs and single molecule measurements. Coupling nanofluidic structures to three-dimensional microfluidic networks is especially powerful and results in applications in sample preconcentration, nanofluidic injection/collection and fast diffusive mixing (160 references).