Neurofilament proteins are known to be transported along axons by slow axonal transport, but the form in which they move is controversial. In previous studies on cultured rat sympathetic neurons, we found that green fluorescent protein-tagged neurofilament proteins move predominantly in the form of filamentous structures, and we proposed that these structures are single-neurofilament polymers. In the present study, we have tested this hypothesis by using a rapid perfusion technique to capture these structures as they move through naturally occurring gaps in the axonal neurofilament array. Because the gaps lack neurofilaments, they permit unambiguous identification of the captured structure. Using quantitative immunofluorescence microscopy and correlative light and electron microscopy, we show that the captured structures are single continuous neurofilament polymers. Thus, neurofilament polymers are one of the cargo structures of slow axonal transport.