Biotinylated dextran amines (BDA) are highly sensitive tools for anterograde and retrograde pathway tracing studies of the nervous system. BDA can be reliably delivered into the nervous system by iontophoretic or pressure injection and visualized with an avidin-biotinylated HRP (ABC) procedure, followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. High molecular weight BDA (10 k) yields sensitive and exquisitely detailed labeling of axons and terminals, while low molecular weight BDA (3 k) yields sensitive and detailed retrograde labeling of neuronal cell bodies. The detail of neuronal cell body labeling can be Golgi-like. BDA tolerates EM fixation and processing well and can, therefore, be readily used in ultrastructural studies. Additionally, BDA can be combined with other anterograde or retrograde tracers (e.g. PHA-L or cholera toxin B fragment) and visualized either by multi-color DAB multiple-labeling - if permanent labels are desired, or by using multiple simultaneous immunofluorescence - if fluorescence viewing is desired. In the same manner, BDA pathway tracing and neurotransmitter immunolabeling can be combined. Note that BDA pathway tracing can also be combined with anterograde or retrograde labeling with fluorescent dextran amines, if one wishes to exclusively use tracers with the favorable transport properties and sensitivities of dextran amines. In this case, the BDA can be visualized together with the fluorescent dextran amines using fluorescence labeling for the BDA, or the fluorescent dextran amines can be visualized together with the BDA by multicolor DAB labeling via immunolabeling of the fluorescent dextran amines using anti-fluorophore antisera. BDA is, thus, a flexible and valuable pathway tracing tool that has gained widespread popularity in recent years.