We analyzed differences in the transendothelial migration (TEM) ability of T-helper (Th)-1 and Th2 cells across a murine endothelial cell line (F-2) under static conditions. The TEM abilities of Th1 cells from mice bearing autoimmune diseases and antigen-specific Th1 cell lines were severalfold higher than those of Th2 cells and lines of the same origin. These preferences were observed without exogenous chemoattractant and were insensitive to pertussis toxin, which completely blocks TEM induced by exogenous chemoattractants. Antibodies against LFA-1 and ICAM-1 as well as CD44 markedly blocked the TEM of Th1 cells. TEM ability was also blocked by pharmacological inhibitors of Src family protein-tyrosine kinases (PP2 and herbimycin A), phosphatidylinositol 3-kinase (wortmannin), and phosphatidylinositol-specific phospholipase C (). Cross-linking of CD44 strongly induced highly elongated morphology in Th1 lines, but weakly in Th2 lines. The pharmacological inhibitors that blocked TEM also inhibited this morphological change, whereas pertussis toxin did not. These data indicate that there are signaling pathways for TEM independent of chemokine attraction, but through adhesion molecules including CD44, and that the preferential TEM ability of Th1 over Th2 cells is formed, at least in part, by intrinsic differences in these pathways.