Acetylcholinesterase was determined histochemically in Sepioteuthis sepioidea nerve fibers observed with the electron microscope. The end product of the reaction appeared focally distributed along the axolemma and attached to its axoplasmic leaflet. At these regions the trilaminar substructure of the axolemma and a narrowing of the axon—Schwann cell interspace were also present. Only random deposits of end product were observed in the axoplasm and in the Schwann cell layer. The material treated with iso-OMPA (an inhibitor of the nonspecific cholinesterase) showed the same exact enzymatic distribution. No positive reaction was observed when eserine or BW 284 C51 (an inhibitor of the acetylcholinesterase) was used. The results show the exact localization of the acetylcholinesterase in the giant nerve fiber of the squid. They also show the possible relationship between the enzyme and sites of special structural arrangement present at the axon-Schwann cell boundary, which might serve as sorts of intercellular junctions between the axon and the Schwann cell.