The velocity of rolling leukocytes is thought to be determined by the expression of adhesion molecules and the prevailing wall shear stress. Here, we investigate whether rapid cleavage of L-selectin may be an additional physiologic regulatory parameter of leukocyte rolling. A unique protease in the membrane of leukocytes cleaves L-selectin after activation, resulting in L-selectin shedding. The hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 completely prevented L-selectin shedding in vitro and significantly decreased the rolling velocity of leukocytes in untreated wild-type C57BL/6 mice from 55 to 35 micrometer/seconds in vivo. When E-selectin was expressed on the endothelium (tumor necrosis factor [TNF]-alpha treatment 2.5-3 h before the experiment), rolling velocity was 4 micrometer/seconds and did not change after the application of KD-IX-73-4. However, KD-IX-73-4 decreased mean rolling velocity by 29% from 23 to 16 micrometer/seconds in E-selectin-deficient mice treated with TNF-alpha. The reduction of velocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a novel method using a local catheter. These results establish a role for L-selectin shedding in regulating leukocyte rolling velocity in vivo.