Human naïve CD4 T cells express low levels of the immunomodulatory receptor CD300a, whereas effector/memory CD4 cells can be either CD300a(+) or CD300a(-). This suggested that CD300a expression could define a specific subset within the effector/memory CD4 T cell subpopulations. In fact, ex vivo analysis of the IFN-gamma producing CD4 T cells showed that they are enriched in the CD300a(+) subset. Moreover, stimulated CD4 T cells producing TNF-alpha and IL-2 besides IFN-gamma (polyfunctional) are predominantly CD300a(+). In addition to producing markedly higher levels of Th1-associated cytokines, the stimulated CD300a(+) CD4 T cells are distinguished by a striking up-regulation of the T-box transcription factor eomesodermin (Eomes), whereas T-bet is up-regulated in both CD300a(+) and CD300a(-) activated CD4 T cells to similar levels. The pleiotropic cytokine TGF-beta1 has a determinant role in dictating the development of this Th1 subset, as its presence inhibits the expression of CD300a and down-regulates the expression of Eomes and IFN-gamma. We conclude that CD300a(+) human Th1 cells tend to be polyfunctional and after stimulation up-regulate Eomes.