In studying reciprocally connected brain networks, it is advantageous to use techniques that allow simultaneous visualization of both efferent and afferent connections from a single injection site. We report on a new technique to achieve this using pressure injections of a mixture of biotinylated dextran amine (BDA) and the beta subunit of cholera toxin (Ctb). Adult male hamsters (n = 12) received 20-30-nl injections of either a 1:1 mixture of BDA (Sigma, 10%) and Ctb (List Biological, 0.5%), or each tracer by itself, into the medial amygdala. Adult female sheep (n = 4) received 200-300 nl of the combined tracer into the A15 region of the hypothalamus. After 1 (hamster) or 2 weeks' (sheep) survival, animals were perfused with 4% paraformaldehyde. Sections were double-labeled, first for BDA histochemistry using nickel-enhanced DAB, then for Ctb using a PAP technique and unenhanced DAB. In all animals, combined injections resulted in clear and consistent patterns of both anterograde and retrograde labeling. Ctb immunoreactivity was distinct and easily distinguished from BDA labeling. There was no evidence for loss of sensitivity of either tracer due to the combined delivery; no differences were seen between combined or single tracer injections in numbers of retrogradely-labeled cells or in the distribution of anterogradely-labeled fibers. In summary, the combined delivery of BDA and Ctb is an easy and reliable technique for simultaneous afferent and efferent tract tracing in both small and large animals; it could potentially be combined with immunocytochemistry to determine the neurochemical content of labeled cells or fibers.