Acetylcholinesterase (AChE) staining in spleens from young adult Sprague-Dawley rats was examined following several denervation paradigms to determine the source of splenic AChE+ nerve fibers. In spleens from all control groups, AChE+ neural-like profiles were present along the vasculature and in the trabeculae. AChE+ reactivity also was present in lymphoid and reticular cells in the spleen, and in neuronal cell bodies in the superior mesenteric-coeliac ganglion (SM-CG). Neurochemical analysis revealed no significant choline acetyltransferase activity in spleens from control animals. Surgical removal of the SM-CG resulted in a total loss of both noradrenergic (NA) and AChE+ nerve profiles, as well as a loss of AChE staining in nonneural compartment in the spleen. On Days 1 and 3 after treatment, chemical sympathectomy with 6-hydroxydopamine also resulted in a loss of both NA and AChE nerve profiles in the spleen, except for a few resistant fibers in the hilar region. AChE reactivity in nonneural compartments also was diminished in chemically denervated regions of the spleen. AChE staining in both neural and nonneural profiles progressively increased from 10 to 56 days after chemical sympathectomy, with a time course and distribution pattern similar to NA fibers reinnervating the spleen. AChE+ staining was preserved following bilateral vagal nerve transection. The miniscule splenic levels of choline acetyltransferase suggest that at best, only a small density of cholinergic nerves distribute to the rat spleen. Further, what cholinergic innervation is present does not arise from the vagus nerve as suggested in the earlier literature. Collectively, the overlapping distribution of AChE+ and NA nerve profiles in spleen and parallel loss of both population of nerve fibers following surgical and chemical sympathectomy support the presence of AChE in NA nerves colocalized with norepinephrine, and thus make AChE+ staining an inappropriate marker for cholinergic innervation in the rat spleen.