Leukocyte accumulation during inflammation depends on the concerted action of selectin and integrin adhesion molecules, which promote capture, rolling, and arrest of these cells on activated endothelium. In addition to interacting with endothelial cells, leukocytes can also adhere to already adherent leukocytes through an L-selectin-dependent mechanism. Initiation of adhesion through this mechanism has been called nucleation and leads to characteristic geometric patterns (ie, clusters and strings) of adherent leukocytes in flow chambers. We have used intravital microscopy of tumor necrosis factor-alpha (TNF-alpha)-treated mouse cremaster muscles to quantitatively investigate the potential role of leukocyte-leukocyte adhesion in initiating and maintaining the leukocyte clusters that are commonly observed in inflamed venules. Our data show that in TNF-alpha-treated venules with diameters between 23 and 108 microm, leukocyte adhesion occurs in clusters that are 19 to 50 microm long and 8 to 44 microm wide. They are almost entirely made up of slow-rolling leukocytes. Of all leukocytes recruited into a cluster (100%), the majority enter the cluster rolling along the endothelium and sharply reduce their velocity in the absence (59%) or presence (15%) of other leukocytes in proximity (one cell diameter). Some of the rolling leukocytes (17%) pass through the cluster without reducing their velocity. Recruitment of leukocytes from the free flow regime into a cluster is a rare event and accounts for only 7 (1.2%) of 476 leukocytes arriving in the cluster. However, of the leukocytes captured from the free flow, 6 initiated contact with a slow-rolling leukocyte rather than making direct contact with the endothelium. Our data show that leukocyte-leukocyte interactions can occur in vivo but are not important for cluster formation. This is confirmed by the observation of normal cluster formation in L-selectin-deficient mice, in which leukocyte-leukocyte interactions under flow are abolished. We conclude that leukocyte-mediated nucleation contributes little to leukocyte recruitment during inflammation in vivo. Cluster formation appears to be dominated by areas of endothelium with a higher expression of E-selectin, because cluster formation is greatly reduced in E-selectin-deficient mice.