Few studies have correlated the occurrence of leukocytes with the time course of ischemia-reperfusion (I-R)-induced tissue injury in skeletal muscle. The goal of this study was to test the hypothesis that leukocytes were responsible for not only the onset, but progression of parenchymal cell injury within skeletal muscle following no-flow ischemia. Thirteen male Wistar rats (150-250 g) were randomly allocated to either a control (no I-R; n = 3), I-R (n = 5), or a leukopenic I-R group (n = 5). Under halothane anesthesia, the extensor digitorum longus muscle was prepared for intravital microscopy to allow video recording of microvascular perfusion and leukocyte flow behavior following 3 hr no-flow ischemia of the hindlimb. Tissue injury was assessed as the ratio of ethidium bromide (impermeant dye)-labeled nuclei to bisbenzimide (permeant dye)-labeled nuclei (E/B). During reperfusion, the I-R group showed a progressive decline in the number of perfused capillaries (NC) (from 19.37 ± 0.04 to 3.34 ± 1.18), while leukopenic and control rats were not significantly different. In the I-R group, the number of rolling leukocytes increased from 4.05 ± 1.93 to 14.77 ± 1.33 at the onset of reperfusion and remained stable throughout the reperfusion period. The number of stuck leukocytes, in the I-R group, progressively increased from 1.41 ± 0.01 prior to ischemia to 4.66 ± 0.01 at the onset of reperfusion to 11.96 ± 0.01 after 90 min. The index of tissue injury (E/B) increased asymptotically from 0.60 ± 0.02 to 0.95 ± 0.01 after 90 min of reperfusion in the I-R group, while leukopenia significantly reduced both the magnitude of tissue injury (i.e., 35% reduction from untreated I-R group) and the onset of such injury. Inspite of the benefit afforded by leukopenia, evidence of tissue injury persisted (20% above control baseline level). We conclude that although leukocytes were responsible for the onset of parenchymal injury in skeletal muscle following 3 hr no-flow ischemia they are not the sole mediators of such injury.