In recent years, the finding of a complete set of molecules related to the cholinergic neurotransmitter system in male gametes of different invertebrate and vertebrate species has opened the question of the possible involvement of this signaling system in the regulation of intracellular ion change, functional to sperm motility and probably also to interaction with the egg. In this work, we localized molecules immunologically related to muscarinic acetylcholine receptors (mAChRs) in sea urchin (Paracentrotus lividus) eggs and zygotes, by use of the monoclonal antibody M35. The ultrastructural analysis of immunoreactivity revealed that the localization of M35-positive molecules is different between unfertilized and fertilized egg. In the unfertilized eggs, immunoreactive molecules were localized inside and associated to the membrane of vesicles, scattered in a thick zone of the cortical cytoplasm. Following fertilization, the oolemma was decored along the surface, and positive vesicles were seldom seen in the cytoplasm. At the surface of fertilized eggs, evident pinocytotic vesicles, recognized by the surrounding coat, indicate that the positive sites correspond to receptorial activity, able to rescue ligand portions. The presence of a cholinergic signaling pattern was also suggested by the presence of molecules immunologically related to acetylcholinesterase (AChE), performing AChE enzyme activity, detected by both immunofluorescent and histochemical methods in membranous organelles belonging to the cortical region of unfertilized eggs.