A study was undertaken to determine the source and terminal distribution of the autonomic nervous system (ANS) innervation of lymphoid tissue in the mediastinum of male and female B10 and Balb/c wildtype and syngeneic nude (nu/nu) mice. Acetylcholinesterase (AChE) histochemistry was used to localize this hydrolytic enzyme in neural tissue and glyoxylic acid fluorescent histochemistry was used to characterize catecholaminergic (CA) innervation. The thymus was innervated by AChE-positive fibers of the vagus, the recurrent laryngeal, and the phrenic nerves. Catecholaminergic innervation was derived from the stellate ganglia and other small ganglia of the thoracic sympathetic nervous system chain. Whereas intrinsic AChE-positive innervation of the thymus was evident at the cortico-medullary boundaries and under the capsule, CA innervation was observed along the trabeculae with perivascular plexuses at the cortico-medullary boundaries and interlobular septa. Free CA fibers were noted in the medulla and in the cortex, where they were often proximal to cortical autofluorescent (CAF) cells. The rudimentary thymus of the adult nude mouse received limited ANS innervation. Only a few CA or AChE-positive fibers were evident, with the majority of fibers associated with the acinar cells of the gland. Mediastinal lymph nodes in male and female wildtype mice varied in their distribution and were frequently found clustered around nerves and ganglia. The intrinsic innervation of lymph nodes by AChE-positive fibers was confined to the subcapsular marginal sinus, with a few vascular-associated fibers in the parenchyma. Catecholaminergic perivascular plexuses were present in the hilar zone, whereas some free fibers were noted under the capsule and in the parenchyma. The mediastinal lymph nodes of male and female nude mice when compared to the lymph nodes of male and female wildtype mouse were found to be increased in number and in distribution. Clusters of these lymph nodes were found in close proximity to ANS tissue. The intrinsic innervation of the nude mediastinal lymph nodes was comparable to that present in the wildtype mouse tissue. The functional implication of these data is discussed with regard to nervous:immune system interactions.