We studied leukocyte interactions in shear flow with peripheral lymph node addressin (PNAd), a mixture of glycoproteins expressed on high endothelial venules (HEV) that is required for lymphocyte homing and has been shown to contain a ligand for L-selectin. T lymphocytes and neutrophils tether and roll on plastic-immobilized PNAd and E-selectin at 1.8 dyn/cm2 wall shear stress, but fail to interact with immobilized ICAM-1, a ligand for LFA-1 and Mac-1, at the same flow rate. Cells roll faster on PNAd than on P-selectin or E-selectin. L-selectin mAb inhibit T lymphocyte and neutrophil tethering to PNAd, but do not inhibit T lymphocyte tethering to purified E-selectin. If allowed to interact with ICAM-1 under static conditions, phorbol ester-treated T lymphocytes, but not resting T lymphocytes, are able to form stationary adhesions that withstand the detachment force generated by 36 dyn/cm2 wall shear stress. In contrast, a wall shear stress of 7.3 dyn/cm2 detaches 50% of resting T lymphocytes bound to PNAd. Incubating T lymphocytes on PNAd and ICAM-1 does not result in adhesion strengthening, suggesting that adhesion through PNAd by L-selectin does not stimulate lymphocyte LFA-1 avidity for ICAM-1. Chemoattractant stimulation of neutrophils or phorbol ester stimulation of lymphoblasts rolling on coimmobilized PNAd and ICAM-1 results in rapid arrest and firm sticking, extending the model of sequential selectin-mediated rolling and subsequent integrin-mediated firm arrest to lymphocytes and ligands expressed on HEV.