This review focuses on the characterization and mechanisms of formation, targeting, and function of extracellular vesicles (EVs), including exosomes and microvesicles. EVs are released by cells and serve as vehicles for the transfer of membrane and cytosolic proteins, lipids, and RNA between cells. The review highlights the challenges in understanding the molecular mechanisms of EV formation and the lack of methods to interfere with cargo packaging or vesicle release, which hinders the identification of their physiological relevance in vivo. It discusses the current state of knowledge on the biogenesis of EVs, including the role of endosomal sorting complexes required for transport (ESCRTs) and other molecular machineries. The review also covers the isolation and characterization of EVs, the molecular composition of EVs, and the mechanisms involved in the release of EVs. Additionally, it explores the interactions of EVs with recipient cells and their functions in physiological and pathological processes, such as immune responses, tumor progression, and neurological disorders. The review emphasizes the need for further research to understand the precise roles of EVs in various biological contexts and the development of standardized methods for EV purification and analysis.This review focuses on the characterization and mechanisms of formation, targeting, and function of extracellular vesicles (EVs), including exosomes and microvesicles. EVs are released by cells and serve as vehicles for the transfer of membrane and cytosolic proteins, lipids, and RNA between cells. The review highlights the challenges in understanding the molecular mechanisms of EV formation and the lack of methods to interfere with cargo packaging or vesicle release, which hinders the identification of their physiological relevance in vivo. It discusses the current state of knowledge on the biogenesis of EVs, including the role of endosomal sorting complexes required for transport (ESCRTs) and other molecular machineries. The review also covers the isolation and characterization of EVs, the molecular composition of EVs, and the mechanisms involved in the release of EVs. Additionally, it explores the interactions of EVs with recipient cells and their functions in physiological and pathological processes, such as immune responses, tumor progression, and neurological disorders. The review emphasizes the need for further research to understand the precise roles of EVs in various biological contexts and the development of standardized methods for EV purification and analysis.