MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by binding to target mRNAs. Recent studies have detected miRNAs in human body fluids, including blood plasma, as extracellular, nuclease-resistant entities. However, the origin and function of these extracellular miRNAs remain unclear. This study confirms that mature miRNAs in blood plasma and cell culture media are highly stable and do not pass through 0.22 μm filters, indicating they are not associated with vesicles. Western blot analysis shows that extracellular miRNAs co-precipitate with Ago2, a component of the RNA-induced silencing complex. This is the first study to show that extracellular miRNAs are predominantly exosome-free and associated with Ago proteins. The authors hypothesize that extracellular miRNAs are by-products of dead cells, stabilized by the Ago2-miRNA complex. While some miRNAs may be associated with exosomes, the majority are not. The study also shows that miRNAs remain stable in extracellular environments for extended periods, suggesting they may serve as biomarkers. The findings highlight the importance of considering Ago2-associated miRNAs in miRNA studies and suggest that exosomal miRNAs may be a small fraction of extracellular miRNAs. The study also discusses the potential of miRNAs as biomarkers for cancer and other diseases, and their role in cell-to-cell communication. The results indicate that miRNAs may be released from cells during apoptosis or necrosis and remain stable in the extracellular space. The study emphasizes the need for standardized protocols in miRNA analysis and the importance of considering the stability of miRNAs when interpreting their biological functions.MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by binding to target mRNAs. Recent studies have detected miRNAs in human body fluids, including blood plasma, as extracellular, nuclease-resistant entities. However, the origin and function of these extracellular miRNAs remain unclear. This study confirms that mature miRNAs in blood plasma and cell culture media are highly stable and do not pass through 0.22 μm filters, indicating they are not associated with vesicles. Western blot analysis shows that extracellular miRNAs co-precipitate with Ago2, a component of the RNA-induced silencing complex. This is the first study to show that extracellular miRNAs are predominantly exosome-free and associated with Ago proteins. The authors hypothesize that extracellular miRNAs are by-products of dead cells, stabilized by the Ago2-miRNA complex. While some miRNAs may be associated with exosomes, the majority are not. The study also shows that miRNAs remain stable in extracellular environments for extended periods, suggesting they may serve as biomarkers. The findings highlight the importance of considering Ago2-associated miRNAs in miRNA studies and suggest that exosomal miRNAs may be a small fraction of extracellular miRNAs. The study also discusses the potential of miRNAs as biomarkers for cancer and other diseases, and their role in cell-to-cell communication. The results indicate that miRNAs may be released from cells during apoptosis or necrosis and remain stable in the extracellular space. The study emphasizes the need for standardized protocols in miRNA analysis and the importance of considering the stability of miRNAs when interpreting their biological functions.