Cytoskeletal polymers and other cytosolic protein complexes are transported along axons in the slow components of axonal transport. Recent studies on the movement of neurofilaments and microtubules in the axons of cultured neurons indicate that these polymers actually move at fast rates and that the movements are also infrequent and highly asynchronous. These observations indicate that the slow rate of slow axonal transport is due to rapid movements interrupted by prolonged pauses, which presents special challenges for studies on the mechanism of movement. This chapter describes the procedures that the author’s laboratory has used to observe and analyze the movement of neurofilaments and microtubules in axons of cultured neurons from the superior cervical ganglia of neonatal rats. In particular, the author describes how to culture these neurons, how to transfect them by nuclear injection, and how to detect the rapid and infrequent movement of cytoskeletal polymers using time-lapse fluorescence imaging.