Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14(++)CD16(-), intermediate CD14(++)CD16(+), and nonclassical CD14(+)CD16(++) monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14(++)CD16(+) monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14(++)CD16(+) monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14(++)CD16(+) monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14(++)CD16(+) monocytes to Ag processing and presentation (eg, CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (eg, TGFB1, AIF1, PTPN6), and to angiogenesis (eg, TIE2, CD105). In conclusion, we provide genetic evidence for a distinct role of CD14(++)CD16(+) monocytes in human immunity. After CD14(++)CD16(+) monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.