The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR) expression and cell lineage. Display omitted •CD161 expression defines specific T cell subsets, including CD8+, CD4+, and TCRγδ+•CD161-expressing lymphocytes possess a conserved transcriptional signature•CD161-expressing lymphocytes display a shared innate response to IL-12+18•CD161 can act as a costimulatory receptor T lymphocytes are conventionally divided into subsets based on expression of coreceptors, cytokines, and surface molecules. Using CyTOF and mRNA microarray analysis, Fergusson et al. identify T lymphocytes that express the C-type lectin CD161 to share a transcriptional profile and innate function across these previously defined subsets.