We investigated the pathogenesis of acute vascular rejection by performing immunofluorescent screening on frozen sections for C1q, C3c, and immunoglobulin M in endomyocardial biopsy specimens from all new heart transplants. Immunofluorescence for C4c, C5, immunoglobulin G, and immunoglobulin A was performed on all positive endomyocardial biopsy specimens. Twenty-eight positive endomyocardial biopsy specimens from six patients were identified, and 22 of those were studied with transmission electron microscopy. Endothelial hyperplasia and myocyte necrosis were prominent in the five female patients with positive immunofluorescence. In addition, macrophages with ultrastructural cytologic features of activation were seen filling capillaries and venules in intimate contact with endothelium and exiting those vessels. Activated macrophages were large cells with abundant cytoplasm and ruffled borders and contained numerous lysosomes, rough endoplasmic reticulum, and mitochondria. Intravascular activated macrophages were identified in five of six patients with positive immunofluorescence but were not seen in any of the endomyocardial biopsy specimens with negative immunofluorescence, including multiple examples of moderate (grades 2 to 3B) and severe (grade 4) acute cellular rejection. In the five female patients with activated macrophages, acute vascular rejection recurred multiple times with one fatality. Review of the files showed three additional, similar cases. The one male patient with positive immunofluorescence but without activated macrophages had only a single episode of acute vascular rejection. Complement and antibodies can activate macrophages, so this finding is not surprising. To the best of our knowledge, this is the first report of the intravascular activation of macrophages, and the first association of this process with acute vascular rejection. Activated macrophages may contribute to myocyte necrosis in acute vascular rejection by compromising blood flow in small vessels.