Vectors derived from the canine adenovirus serotype 2 (CAV-2) possess a high neurotropism and efficient retrograde transport that lead to widespread neuronal transduction in the central nervous system (CNS) of various animals. These abilities are due to the engagement of virions to the coxsackievirus and adenovirus receptor at the surface of neurons, which is linked to the endocytic and axonal transport machineries. The trafficking of CAV-2 and the coxsackievirus and adenovirus receptor (CAR) can be visualized ex vivo by incubating primary neurons (e.g., motoneurons and hippocampal neurons) with fluorescently labeled virions or recombinant viral proteins. Using this approach, we could recapitulate the mechanisms responsible for long-range transport of adenovirus in neurons.