Methods to define circuit organization in the brain are largely based upon the axonal transport capabilities of neurons. Numerous tracers have been developed since the 1970s that are sequestered by neurons and transported through axons in either the anterograde or retrograde directions. These tracers have been widely applied and are integral to the literature that has defined the functional organization of the nervous system. Nevertheless, with few exceptions these tracers do not cross synapses and therefore cannot provide insight into the polysynaptic organization of neurons devoted to a particular function. Viral transneuronal tracers introduced in the 1980s have been developed to fill this void. These tracers exploit the abilities of neurotropic viruses to invade neurons and generate infectious progeny that cross synapses to infect other neurons within a circuit. The method has become increasingly popular with the development of recombinant strains of virus that are reduced in virulence and express unique reporters. In this chapter we provide an overview of guidelines for the use of pseudorabies virus for transneuronal tracing.