Biocytin, a naturally occurring low molecular weight analog of biotin, was evaluated as a neuroanatomical tract-tracing marker in the adult rat brain. Since it retains high-affinity binding to avidin, biocytin can be labelled with avidinylated visualization reagents. Iontophoretic or pressure injections resulted in filling of cell bodies and dendrites around the injection site and their efferent axonal processes and boutons. Retrogradely labelled neurons were occasionally observed at a distance but only with large injections. Anterograde tracing with biocytin is successful even in animals that are quite old, in contrast to lectins and HRP conjugates, and offers advantages in delivery, tissue processing, selection of light and/or electron microscopic labels, time to obtain results, and cost over many conventional tracers.