Fluorescent dextran amines have recently been reported to be useful for anterograde pathway tracing. However, fluorescent markers are not always ideal for detailed mapping studies. We therefore evaluated the efficacy of a biotinylated dextran amine (BDA) for anterograde labeling in several different preparations. BDA was visualized with an avidin-biotinylated HRP (ABC) procedure followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. After iontophoretic injections of BDA into neocortex-like telencephalic regions in pigeons or into visual or somatosensory cortex in rats, there was excellent and abundant labeling of axons and terminals in forebrain, midbrain and hindbrain target areas with 1-week survival times. Large pressure injections of BDA into the avian telencephalon were also found to result in extensive anterograde labeling. We then carried out a series of studies using 2-color DAB double-labeling to determine effective approaches for combining BDA labeling with other labeling methods. Using an isolated embryonic chick spinal cord-hindlimb preparation, we combined BDA labeling with another anterograde labeling method to differentially label two sets of projections. In these studies, sensory neuron and motoneuron projections into the limb from the same segmental level, or motoneuron projections into the limb from two separate segments were differentially labeled by using HRP (visualized first with a blue/black metal-DAB reaction) and BDA (visualized second with a brown DAB reaction). In other double-labeling studies, we combined BDA labeling of axons and terminals with immunohistochemical labeling of neurons. In these experiments, telencephalic neurons in pigeons or rats were labeled immunohistochemically for parvalbumin or substance P (using a brown DAB reaction) and BDA-labeled axons were labeled blue/black (using a metal-intensified DAB reaction). Double-labeling was successful regardless of whether the entire immunohistochemical labeling procedure preceded or followed the BDA labeling procedure. Together, these studies show that BDA is effective for anterograde pathway tracing and can be used in double-label studies with other labeling methods.