Previous studies of myocardial ischemia suggest that complement activation may play a central role in the inflammatory response during reperfusion. Our previous work has demonstrated neutrophil chemotactic activity to be present in reperfusion canine cardiac lymph after myocardial ischemia and infarction. To evaluate the contribution of the complement-dependent anaphylatoxin C5a to this neutrophil chemotactic activity, rabbit antiserum to canine C5a was prepared. At dilutions >1:500 but <1:2,000, the antiserum abolished the ability of zymosan-activated dog serum to induce a ruffled, bipolar morphology in isolated neutrophils used as a bioassay of chemotactic stimulation. This antiserum did not affect similar morphological changes in neutrophils exposed to platelet activating factor (10−7–10−6M) or recombinant human interleukin-8 (10−9–10−8M); thus, we deemed it functionally specific for canine C5a. In a pattern similar to what we previously reported, cardiac lymph collected before a 1-hour ligation of the left circumflex coronary artery had little ability to alter the morphology of canine neutrophils (shape change index, 11.3±4.6, mean±SEM; n=7), but by 1 hour of reperfusion, lymph activated neutrophils significantly in five of seven dogs (mean shape change index, 72.6±17.7; p<0.01). At 2 hours of reperfusion, neutrophil activation by lymph occurred in six of seven dogs (mean shape change index, 103.1±22.2). At 3 hours of reperfusion, cardiac lymph of only three of six dogs caused neutrophil activation, and at 4 hours of reperfusion, this activity was evident in lymph from only two of five dogs. In all cases, however, the neutrophil stimulatory activity of cardiac lymph was inhibited 85–90% by addition of anti-C5a (p<0.01). Preimmunization serum had no effect. Thus, these data indicate that C5a is the predominant chemotactic factor in the first 4 hours after reperfusion of ischemic myocardium in the dog.