The role of leukocytes and nonleukocyte-derived reactive oxygen metabolites (ROMs) in reperfusion-induced skeletal muscle injury was determined. Male rats received 2 h no-flow hindlimb ischemia-reperfusion (I/R, n = 6) or were rendered neutropenic via antineutrophil serum (ANS) before I/R (I/R + ANS, n = 5). Oxygen radicals in the absence of neutrophils were tested by administration of dimethylthiourea (DMTU) (I/R + ANS + DMTU, n = 5). Perfused capillaries (CD(per)) and rolling (L(r)), adherent (L(a)), and extravasated leukocytes (L(e)) in the extensor digitorum longus muscle were measured every 15 min during 90 min of reperfusion using intravital microscopy. The vital dyes bisbenzimide (BB) and ethidium bromide (EB) provided direct measures of tissue injury (EB/BB). CD(per) decreased immediately on reperfusion in the I/R and I/R + ANS groups. CD(per) in the I/R + ANS + DMTU group remained at baseline throughout reperfusion. L(a) increased in the I/R group; however, EB/BB was the same between I/R and I/R + ANS groups. Injury in the I/R + ANS + DMTU group did not differ from other groups > or =60 min, after which EB/BB became significantly lower. L(e) did not differ between groups and was highly correlated to tissue injury. The results suggest that L(e) lead to parenchymal injury, and ROMs lead to perfusion deficits during the early reperfusion period after ischemia.