This study investigates the stability, origin, and function of extracellular circulating microRNAs (miRNAs) in human body fluids and cell culture media. The authors confirmed that mature miRNAs are highly stable in blood plasma and cell culture media, and most miRNAs completely passed through 0.22 μm filters but remained in the supernatant after ultracentrifugation at 110,000 g, indicating their non-vesicular origin. Western blot immunoassay revealed that extracellular miRNAs ultrafiltrated together with the 96 kDa Ago2 protein, a component of the RNA-induced silencing complex. Co-immunoprecipitation experiments showed that miRNAs in both blood plasma and cell culture media co-immunoprecipitated with anti-Ago2 antibody in a detergent-free environment. This is the first study to demonstrate that extracellular miRNAs are predominantly exosome-free and associated with Ago proteins. The authors hypothesize that extracellular miRNAs are mostly by-products of dead cells that persist in the extracellular space due to the high stability of the Ago2 protein and the Ago2-miRNA complex. However, the study does not rule out the possibility that some miRNAs can be associated with exosomes. The findings suggest that the majority of circulating miRNAs in blood plasma and cell culture media are not exosome-associated, and that the stability of miRNA/Ago complexes may play a crucial role in their persistence in the extracellular environment.This study investigates the stability, origin, and function of extracellular circulating microRNAs (miRNAs) in human body fluids and cell culture media. The authors confirmed that mature miRNAs are highly stable in blood plasma and cell culture media, and most miRNAs completely passed through 0.22 μm filters but remained in the supernatant after ultracentrifugation at 110,000 g, indicating their non-vesicular origin. Western blot immunoassay revealed that extracellular miRNAs ultrafiltrated together with the 96 kDa Ago2 protein, a component of the RNA-induced silencing complex. Co-immunoprecipitation experiments showed that miRNAs in both blood plasma and cell culture media co-immunoprecipitated with anti-Ago2 antibody in a detergent-free environment. This is the first study to demonstrate that extracellular miRNAs are predominantly exosome-free and associated with Ago proteins. The authors hypothesize that extracellular miRNAs are mostly by-products of dead cells that persist in the extracellular space due to the high stability of the Ago2 protein and the Ago2-miRNA complex. However, the study does not rule out the possibility that some miRNAs can be associated with exosomes. The findings suggest that the majority of circulating miRNAs in blood plasma and cell culture media are not exosome-associated, and that the stability of miRNA/Ago complexes may play a crucial role in their persistence in the extracellular environment.