Acetylcholinesterase (AChE) was long thought to be an enzyme found specifically at the sites of nerve synapses and neuromuscular junctions. It has also been found to occur, however, in cells that are not involved with neurotransmission. This study presents the ultrastructural localization of AChE activity in human thymus cells, using the indirect thiocholine method. Cytochemical demonstration of the enzyme was based on the coupling of acetylthiocholine iodide hydrolysis to the precipitation of heavy metal salts. AChE activity was selectively revealed in the perinuclear cisternae, within the endoplasmic reticulum, and in the Golgi complex of thymic lymphocytes and epithelial cells. Evidence of the presence of reaction product in the latter cells was also found in vesicles that opened into the extracellular space. This is the first demonstration of AChE in human thymus cells. Its possible physiologic role in the thymus gland is discussed.