Extracellular vesicles (EVs) are crucial for intercellular communication, transferring functional proteins, lipids, and RNAs. EVs include microvesicles, ectosomes, shedding vesicles, microparticles, and exosomes, with exosomes originating from endosomal multivesicular bodies (MVBs). Exosomes are involved in various physiological and pathological processes, and their potential as biomarkers and therapeutic agents is growing. Tetraspanins, a protein superfamily, are abundant in EVs and play key roles in their biogenesis, targeting, and function. They organize tetraspanin-enriched microdomains (TEMs), which are functional membrane microdomains that regulate protein sorting and signaling. Tetraspanins have four transmembrane domains and are involved in cell adhesion, motility, and signaling. Their extracellular and transmembrane domains are crucial for their function, with the extracellular domain containing conserved cysteine residues and the transmembrane domain involved in protein folding and interactions. The cytoplasmic domain contains sorting signals that direct tetraspanins to specific intracellular compartments. Tetraspanins interact with various proteins, including integrins, IgSF members, and signaling molecules, and are involved in the formation of TEMs. These microdomains are essential for the sorting and targeting of proteins to EVs and for the function of EVs in intercellular communication. Tetraspanins are important exosome markers, with CD9, CD63, CD81, CD82, and CD37 being particularly enriched in exosomes. They are used to identify and assess the purity of exosomes. Tetraspanins are involved in exosome biogenesis, with the ESCRT machinery playing a role in their secretion. However, some exosome secretion can occur independently of ESCRTs, with tetraspanins playing a fundamental role in this process. Tetraspanins also influence the cargo selection of EVs, including the sorting of proteins and RNAs into EVs. Tetraspanins are involved in the targeting and uptake of EVs by recipient cells, with their interactions with adhesion molecules and proteases influencing this process. They also play a role in antigen presentation by EVs, with tetraspanins organizing MHC complexes and facilitating the presentation of antigens to T cells. Tetraspanins are important in immune responses, with their involvement in the formation of the immunological synapse and the activation of T cells. Their role in antigen presentation by EVs highlights their potential in immunotherapy and the development of cell-free vaccines. Overall, tetraspanins are essential for the function of EVs in intercellular communication, disease progression, and therapeutic applications.Extracellular vesicles (EVs) are crucial for intercellular communication, transferring functional proteins, lipids, and RNAs. EVs include microvesicles, ectosomes, shedding vesicles, microparticles, and exosomes, with exosomes originating from endosomal multivesicular bodies (MVBs). Exosomes are involved in various physiological and pathological processes, and their potential as biomarkers and therapeutic agents is growing. Tetraspanins, a protein superfamily, are abundant in EVs and play key roles in their biogenesis, targeting, and function. They organize tetraspanin-enriched microdomains (TEMs), which are functional membrane microdomains that regulate protein sorting and signaling. Tetraspanins have four transmembrane domains and are involved in cell adhesion, motility, and signaling. Their extracellular and transmembrane domains are crucial for their function, with the extracellular domain containing conserved cysteine residues and the transmembrane domain involved in protein folding and interactions. The cytoplasmic domain contains sorting signals that direct tetraspanins to specific intracellular compartments. Tetraspanins interact with various proteins, including integrins, IgSF members, and signaling molecules, and are involved in the formation of TEMs. These microdomains are essential for the sorting and targeting of proteins to EVs and for the function of EVs in intercellular communication. Tetraspanins are important exosome markers, with CD9, CD63, CD81, CD82, and CD37 being particularly enriched in exosomes. They are used to identify and assess the purity of exosomes. Tetraspanins are involved in exosome biogenesis, with the ESCRT machinery playing a role in their secretion. However, some exosome secretion can occur independently of ESCRTs, with tetraspanins playing a fundamental role in this process. Tetraspanins also influence the cargo selection of EVs, including the sorting of proteins and RNAs into EVs. Tetraspanins are involved in the targeting and uptake of EVs by recipient cells, with their interactions with adhesion molecules and proteases influencing this process. They also play a role in antigen presentation by EVs, with tetraspanins organizing MHC complexes and facilitating the presentation of antigens to T cells. Tetraspanins are important in immune responses, with their involvement in the formation of the immunological synapse and the activation of T cells. Their role in antigen presentation by EVs highlights their potential in immunotherapy and the development of cell-free vaccines. Overall, tetraspanins are essential for the function of EVs in intercellular communication, disease progression, and therapeutic applications.