The distribution of cholinesterase activity in the human retina was evaluated using histochemical methods. The butyrylcholine esterase (BuChE) inhibitor tetraisopropylpyrophosphoramide was used to localize acetylcholinesterase (AChE) activity, the AChE inhibitor BW284c51 was used to localize BuChE activity, and eserine was used to inhibit all cholinesterase activity in control incubations. We have found specific AChE activity in the inner plexiform layer (IPL) and BuChE activity is apparently associated with blood vessels at the light microscopic level. At the electron microscopic level, AChE reaction product is seen both at apparently synaptic and manifestly non-synaptic sites. Reaction product is found adjacent to neural processes in the IPL; these processes have an ultrastructure characteristic of amacrine cells. Although reaction product is seen adjacent to bipolar, ganglion and Müller cells, it is only present where they come into contact with apparent amacrine cell profiles. Using longer incubations, reaction product is found in the perikarya of cells with the morphology and position of amacrine and displaced amacrine cells. There is also some evidence for AChE activity adjacent to lateral processes at a rare type of rod synapse in the outer plexiform layer. The observations reported here strengthen the evidence for cholinergic neurotransmission in the human retina.