This study examined the effects of chlorpromazine on the expression of CD81 in representative human cell lines of the central nervous system origin. Chlorpromazine is a neuroleptic that acts as a strong antagonist of dopamine receptors, which participate in processes like learning, memory, motivation, pleasure etc. Also, chlorpromazine affects many other membrane and cytosolic proteins, resulting in decreased communication between neurons and glial cells, especially astrocytes. Nevertheless, it has not yet been determined if chlorpromazine affects a specific form of intercellular communication that involves extracellular vesicles (EVs). EVs are membranous nanostructures that carry proteins, nucleic acids, lipids and other molecules. All types of cells release EVs and with them secretory cells can change the phenotype of recipient cells. The CD81 molecule is a member of the tetraspanin protein family ubiquitously expressed in all tissues, including the central nervous system. Next to the expression in plasma membrane, CD81 is present in intracellular membrane compartments and it is a recognized marker of EVs. In the nervous system, this protein is important for neuron-astrocyte interactions and is implicated in the response of astrocytes and microglia to trauma and disease. In this study, the human glioblastoma cell line U-87 MG and neuroblastoma SH-SY5Y cells were treated with increasing concentrations of chlorpromazine (4, 8, 16 and 32 µg/ml) for 24 hours. Flow cytometry analysis of the CD81 expression has shown that chlorpromazine affects the examined cell lines in different ways. U-87 MG cells responded to chlorpromazine with a dose-dependent increase of CD81 protein level, while SH-SY5Y cells showed decreased CD81 levels. These findings are the first to date that associate chlorpromazine with changes in CD81 expression. Considering the role of this protein in neuron-astrocyte interactions and its presence in EVs, future studies are necessary to examine the effects of chlorpromazine treatment on processes important for intercellular communication in the nervous system.