Although there is increasing evidence that mismatched donor HLA antigens are associated with a lowering of survival of human cardiac allografts, the effect of antibodies that bind those antigens is less clear. The existence of lymphocytotoxic antibodies prior to cardiac transplantation has been associated with a poor outcome in the majority of reports of relevant studies, as has their appearance post-transplantation. But how such antibodies, especially those with HLA specificity, cause poor outcomes has been poorly understood. The purpose of this study was to investigate the effect of anti-HLA antibodies appearing in the circulation after human orthotopic heart transplantation. Such antibodies were identified by a standard microlymphocytotoxicity technique using panels of frozen lymphocytes from normal donors who had been tissue typed. Of 74 patients transplanted over a 12-month period, 4 (5.4%) developed alloantibodies specific for mismatched donor HLA antigens. The first patient developed antibodies to HLA-A23 and B44 together with poor ventricular function and vascular rejection requiring retransplantation within 4 months. The other patients (3) developed antibodies specific for HLA-DQ antigens and experienced variable numbers of episodes of cellular rejection with no evidence of vascular rejection on endomyocardial biopsy. Two of these three patients died (8 and 11 months post-transplant) after three and six rejection episodes, respectively. The one surviving patient had seven rejection episodes and continues to have poor ventricular function 18 months post-transplant. We conclude that alloantibodies specific for mismatched donor HLA antigens may have a deleterious effect on the outcome of the human cardiac allograft and should be monitored closely post-transplant. Furthermore, such antibodies may mediate effects on the transplanted heart which are not detectable in specimens obtained by endomyocardial biopsy.