This study characterized human plasma-derived exosomal RNAs using deep sequencing. The researchers analyzed three human plasma samples and evaluated three small RNA library preparation protocols from different manufacturers. They generated 14 sequencing libraries (7 replicates) and obtained a total of 101.8 million raw single-end reads, with an average of about 7.27 million reads per library. Sequence analysis revealed that microRNAs (miRNAs) were the most abundant RNA species, accounting for over 42.32% of all raw reads and 76.20% of all mappable reads. A total of 593 miRNAs were detectable, with the five most common miRNAs collectively accounting for 48.99% of all mappable miRNA sequences. The miRNA target gene enrichment analysis suggested that these miRNAs may play important roles in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. Other RNA species, including ribosomal RNA, long non-coding RNA, piwi-interacting RNA, transfer RNA, small nuclear RNA, and small nucleolar RNA, were also detected. The three tested commercial kits generated sufficient DNA fragments for sequencing but each had significant bias toward capturing specific RNAs. This study demonstrated that a wide variety of RNA species are embedded in circulating vesicles and that this is the first report applying deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.This study characterized human plasma-derived exosomal RNAs using deep sequencing. The researchers analyzed three human plasma samples and evaluated three small RNA library preparation protocols from different manufacturers. They generated 14 sequencing libraries (7 replicates) and obtained a total of 101.8 million raw single-end reads, with an average of about 7.27 million reads per library. Sequence analysis revealed that microRNAs (miRNAs) were the most abundant RNA species, accounting for over 42.32% of all raw reads and 76.20% of all mappable reads. A total of 593 miRNAs were detectable, with the five most common miRNAs collectively accounting for 48.99% of all mappable miRNA sequences. The miRNA target gene enrichment analysis suggested that these miRNAs may play important roles in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. Other RNA species, including ribosomal RNA, long non-coding RNA, piwi-interacting RNA, transfer RNA, small nuclear RNA, and small nucleolar RNA, were also detected. The three tested commercial kits generated sufficient DNA fragments for sequencing but each had significant bias toward capturing specific RNAs. This study demonstrated that a wide variety of RNA species are embedded in circulating vesicles and that this is the first report applying deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.