Localization of circulating lymphocytes to a site of inflammation is paramount for the development and maintenance of an immune response. In vitro studies using cell lines have previously demonstrated that rolling and adhesion of lymphocytes on endothelium requires CD44 interactions with hyaluronan (HA). To date, whether CD44 has a role in mediating CD4(+)-polarized T-helper 1 (Th1) and Th2 lymphocyte interactions with the endothelium in vivo is yet to be determined. In this study we used intravital microscopy to demonstrate that both Th1 and Th2 lymphocytes use CD44 to roll and adhere to tumor necrosis factor-alpha (TNFalpha)-activated microvasculature. Furthermore, chimeric studies imply that CD44 expression by both the endothelium and lymphocytes is essential for these interactions to occur. HA was also necessary for T cell-endothelial cell interactions in vivo and Th1 and Th2 cells rolled on immobilized HA in vitro via CD44. In vitro, both Th1 and Th2 lymphocytes have increased expression of CD44 and greater binding of fluorescent HA than naive cells. The interactions of Th1 and Th2 cells were entirely dependent upon both P-selectin and CD44 in vivo, but did not appear to be counter ligands in vitro. Taken together, these results suggest that CD44 and HA are key to both Th1 and Th2 lymphocyte interactions with the TNFalpha-activated endothelium and raises the possibility of cooperativity between the P-selectin/PSGL-1 and HA/CD44 pathways for Th1 and Th2 rolling in vivo.