The widespread use of new axonal transport tracing techniques in the ANS has resulted in substantially revised and amended descriptions of ANS organization. The present review suggests, however, that at least some of the results on which proposed revisions of ANS anatomy have been based have incorporated artifacts and therefore should be cautiously interpreted. The peripheral nervous system and viscera are composed in part of connective and endothelial tissues that are porous or 'leaky' to solutes with appropriate chemical characteristics, including the major tracer compounds. As a result, several extra-axonal routes for redistribution of label from the application site into other tissues are present. These include (1) diffusion through tissue membranes to enter directly adjacent tissues and (2) leakage into extracellular fluids within the body cavity, vasculature, lymphatics, exocrine ducts, or organ lumens to migrate to more distant tissues. As a consequence of the extreme sensitivity of the methods used, such redistribution of even minute amounts of label can produce false positives. Review of autonomic neuroanatomy suggests additional mechanisms, including tracer uptake by fibers of passage, can produce artifactual staining. Based on these surveys of tissue composition, tracer characteristics and sources of artifact, experimental controls and criteria for identifying and avoiding labeling artifacts are described. Since no single procedure is foolproof for ANS experimentation, the routine application of multiple controls, particularly ones which restrict or prevent tracer diffusion, are needed.