Background: Immunofluorescence staining of endomyocardial biopsy (EMB) specimens to detect the complement fragment C4d is used to diagnose antibody-mediated rejection. However, data are limited regarding the utility of routine staining for C4d in clinical care. Methods: This study retrospectively reviewed the clinical course of adult cardiac transplant recipients who underwent ≥ 2 EMBs with immunofluorescence C4d staining at the University of Texas Southwestern Medical Center since September 2006. C4d staining was performed by the immunohistochemistry laboratory and interpreted by the members of the surgical pathology department, in conjunction with interpretation of the routine hematoxylin and eosin staining. Donor-specific antibodies (DSA) were routinely assessed at the time of clinical rejection. Results: Of 67 patients, specimens were positive for C4d (C4d+) in 14 and negative for C4d (C4d-) in 53. The frequency of acute cellular rejection (ACR) in these 2 groups was 57% (8 of 14, designated C4d+/ACR+) vs 11% (6 of 53, designated C4d-/ACR+; p < 0.001). Significantly more patients with a positive C4d specimen had a positive retrospective donor specific crossmatch, presence of DSA after transplantation, and depressed graft function (p < 0.01 for each). Conclusions: Positive C4d immunofluorescence staining on EMB specimens was associated with ACR, reduced allograft function, a positive retrospective crossmatch, and the presence of DSA after transplantation. The latter 2 observations support the contention that C4d deposition is a marker of antibody-mediated rejection. Routine evaluation of C4d staining is feasible in the clinical setting and may identify variable patterns of rejection.