IL-17-producing Th cells (Th17) are a distinct subset of effector cells that bridge the innate and adaptive immune system and are implicated in autoimmune disease processes. CD4(+) splenocytes from DO11.10 mice were activated with OVA peptide(323-339) and maintained under Th17 polarization conditions, resulting in significantly higher proportions of IL-17(+) T cells compared with nonpolarized (Th0) cells. Th17-polarizing conditions significantly increased the proportion of cells expressing the chemokine receptors CCR2, CCR6, and CCR9 when compared with Th0 cells. In contrast, there was a significant decrease in the proportion of cells expressing CXCR3 under Th17-polarizing conditions compared with nonpolarizing conditions. The respective chemokine agonists for CCR2 (CCL2 and CCL12), CCR6 (CCL20), and CCR9 (CCL25) elicited migration and PI-3K-dependent signaling events in Th17-polarized cells, thus indicating that all three receptors were functionally and biochemically responsive. Furthermore, postmigration phenotypic analysis demonstrated that the agonists for CCR2 and CCR6, but not CCR9, stimulated a modest enrichment of IL-17(+) cells compared with the premigration population. Pan-isoform inhibitors of PI-3K/Akt signaling prevented CCR2- and CCR6-mediated, polarized Th17 cell migration in a concentration-dependent manner. The unique chemokine receptor expression pattern of Th17 cells and their corresponding PI-3K-dependent migratory responses are important for understanding the pathogenesis of autoimmune diseases and may provide opportunities for the application of CCR2 and CCR6 antagonists and PI-3K isoform-selective inhibitors in defined inflammatory settings.