Small ( approximately 15 nm diameter), highly fluorescent conjugated polymer nanoparticles were evaluated for nanoscale 2D and 3D tracking applications. Nanoparticles composed of conjugated polymers possess high absorption cross sections, high radiative rates, and low or moderate aggregation quenching, resulting in extraordinarily high fluorescent brightness. The bright fluorescence ( approximately 200 000 photons detected per particle per 20 ms exposure) yields a theoretical particle tracking uncertainty of less than 1 nm. A lateral tracking uncertainty of 1-2 nm was determined from analysis of trajectories of fixed and freely diffusing particles. Axial (Z) position information for 3D particle tracking was obtained by defocused imaging. Nanoscale tracking of single particles in fixed cells was demonstrated, and a range of complex behaviors, possibly due to binding/unbinding dynamics, were observed.