Extracellular vesicles (EVs) are membrane-bound nanoscale particles released by all prokaryotic and eukaryotic cells, carrying proteins, lipids, RNA, and DNA. They facilitate intercellular communication by delivering information between cells, with evidence of functional effects on recipient cells. EVs contain various RNA biotypes, including small non-coding RNAs, fragmented and intact mRNAs, rRNAs, and lncRNAs. These RNAs can influence recipient cell gene expression and function. EVs have attracted interest due to their potential clinical utility as biomarkers and therapeutic vehicles. However, many aspects of EV biology remain unclear, including selective cargo loading, heterogeneity in size and composition, and mechanisms of uptake and cargo fate. Understanding the biogenesis, transport, and function of EV RNAs is crucial for their clinical application. EVs can deliver RNAs to recipient cells, with examples including cardiac repair, neurodegeneration, and cancer progression. The RNA content of EVs reflects the source cell's type and physiological state but differs significantly from the source cell. EVs are heterogeneous, with subclasses such as exosomes, microvesicles, large oncosomes, and apoptotic bodies. Challenges include distinguishing EV subclasses, standardizing terminology, and understanding RNA delivery mechanisms. EVs can serve as RNA carriers, with RNA types categorized into functional, predicted, and unknown. RNA packaging into EVs involves various mechanisms, including specific RNA sequences, membrane lipid interactions, and RBPs. EVs can deliver functional RNA to recipient cells, but endosomal escape is critical for RNA function. EVs can be internalized via endocytosis, macropinocytosis, or direct membrane fusion. RNA delivery to recipient cells is influenced by the cell type and physiological state. EVs can also trigger immune responses, with some RNAs acting as pathogen-associated molecular patterns. EVs have potential applications in disease detection and treatment, including cardiovascular, neurodegenerative, and metabolic diseases, as well as cancers. However, challenges remain in understanding the mechanisms of RNA delivery, cargo fate, and immune responses. Further research is needed to overcome these challenges and fully harness the potential of EVs in clinical applications.Extracellular vesicles (EVs) are membrane-bound nanoscale particles released by all prokaryotic and eukaryotic cells, carrying proteins, lipids, RNA, and DNA. They facilitate intercellular communication by delivering information between cells, with evidence of functional effects on recipient cells. EVs contain various RNA biotypes, including small non-coding RNAs, fragmented and intact mRNAs, rRNAs, and lncRNAs. These RNAs can influence recipient cell gene expression and function. EVs have attracted interest due to their potential clinical utility as biomarkers and therapeutic vehicles. However, many aspects of EV biology remain unclear, including selective cargo loading, heterogeneity in size and composition, and mechanisms of uptake and cargo fate. Understanding the biogenesis, transport, and function of EV RNAs is crucial for their clinical application. EVs can deliver RNAs to recipient cells, with examples including cardiac repair, neurodegeneration, and cancer progression. The RNA content of EVs reflects the source cell's type and physiological state but differs significantly from the source cell. EVs are heterogeneous, with subclasses such as exosomes, microvesicles, large oncosomes, and apoptotic bodies. Challenges include distinguishing EV subclasses, standardizing terminology, and understanding RNA delivery mechanisms. EVs can serve as RNA carriers, with RNA types categorized into functional, predicted, and unknown. RNA packaging into EVs involves various mechanisms, including specific RNA sequences, membrane lipid interactions, and RBPs. EVs can deliver functional RNA to recipient cells, but endosomal escape is critical for RNA function. EVs can be internalized via endocytosis, macropinocytosis, or direct membrane fusion. RNA delivery to recipient cells is influenced by the cell type and physiological state. EVs can also trigger immune responses, with some RNAs acting as pathogen-associated molecular patterns. EVs have potential applications in disease detection and treatment, including cardiovascular, neurodegenerative, and metabolic diseases, as well as cancers. However, challenges remain in understanding the mechanisms of RNA delivery, cargo fate, and immune responses. Further research is needed to overcome these challenges and fully harness the potential of EVs in clinical applications.