Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E(2) (PGE(2)) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE(2), peripheral blood mononuclear cells produced higher interleukin-17 (IL-17), C-C chemokine ligand 20 (CCL20)/macrophage inflammatory protein 3alpha (MIP-3alpha), CXC chemokine ligand 8 (CXCL8)/IL-8, and lower interferon-gamma and IL-22 levels than in control cultures. Exogenous PGE(2) and IL-23 synergized in inducing IL-17, whereas indomethacin and IL-23 blockade drastically reduced IL-17 but not interferon-gamma production. Furthermore, IL-1 but not tumor necrosis factor was absolutely required for IL-17 production. PGE(2) doubled the frequency of CD4+ T cells producing IL-17 and within the CD4+ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4+ T-cell lines, the production of IL-17 segregated with the CCR6+ subset. In the presence of CCR6+ compared with CXCR3+ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and IL-1beta. These results identify PGE(2) and IL-23 as participating in the expansion of CD4+ T cells endowed with high IL-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.