Abstract Extravasation of pro-inflammatory T cells is critical for their effectiveness in host defense and for mediating tissue damage in immune-mediated disease. These cells co-express multiple chemokine receptors and understanding the activities of individual receptors in extravasation is essential for exploiting the chemokine system for therapeutic benefit – either to block or enhance the trafficking of these cells into sites of inflammation. CCR6 is expressed on all human Th17 cells, and we found that CCR6 +CCR2 +cells are enriched both in cells that express a pathogenic Th17 signature, including the ability to produce GM-CSF and IFNγ, and are unusually efficient at transendothelial migration (TEM). Ligands for CCR6, CCR5 and CXCR3 were secreted by and bound to endothelial cells activated with TNFα and IFNγ, and these receptors could all mediate firm arrest of CCR6 +CCR2 +cells under flow - but could not mediate TEM. TEM required CCR2, whose ligands, CCL2, CCL7 and CCL8, were all secreted by activated endothelial cells but failed to bind to endothelial cell surfaces. Chemokines were secreted from the basal as well as the apical sides of endothelial cells. Pre-treating CCR6 +CCR2 +cells with CCL2 or transducing endothelial cells to express a surface-bound CCL2-CXCL9 chimera led to both CCR2-mediated arrest and, importantly, inhibition of TEM. Taken together, our data suggest that while arrest requires endothelial surface-bound chemokine, TEM requires a transendothelial gradient maintained by the rapid dilution of non-bound luminal chemokine by flow. Consequently, mediating arrest versus TEM are not inherent, but nonetheless mutually exclusive activities of receptors that depend, respectively, on binding vs. non-binding chemokines. NIH