We have developed a novel pulse-escape fluorescence photoactivation technique to investigate the long-term pausing behavior of axonal neurofilaments. Cultured sympathetic neurons expressing a photoactivatable green fluorescent neurofilament fusion protein were illuminated with violet light in a short segment of axon to create a pulse of fluorescent neurofilaments. Neurofilaments departed from the photoactivated regions at rapid velocities, but the overall loss of fluorescence was slow because many of the neurofilaments paused for long periods of time before moving. The frequency of neurofilament departure was more rapid initially and slower at later times, resulting in biphasic decay kinetics. By computational simulation of the kinetics, we show that the neurofilaments switched between two distinct states: a mobile state characterized by intermittent movements and short pauses (average = 30 s) and a stationary state characterized by remarkably long pauses (average = 60 min). On average, the neurofilaments spent 92% of their time in the stationary state. Combining short and long pauses, they paused for 97% of the time, resulting in an average transport rate of 0.5 mm/d. We speculate that the relative proportion of the time that neurofilaments spend in the stationary state may be a principal determinant of their transport rate and distribution along axons, and a potential target of mechanisms that lead to abnormal neurofilament accumulations in disease.