Background Rejection of transplanted organs is caused by alloimmune responses, primarily against HLA molecules. Anti-donor HLA antibodies are associated with antibody-mediated rejection (AMR) and poor graft outcome. Because of clinical interest in detecting these antibodies, new technologies have recently been introduced to increase the sensitivity of detection. The Luminex Single-Antigen (LSA) bead assay may yield new information, but it must be validated against biological and clinical data. Material and Methods Based on previously published data regarding the in vitro effects of anti-HLA antibodies on lymphocytes, we measured the effect on lymphocytes of sera from patients on the transplant waiting list who had high titers of anti-HLA antibodies. Anti-CD3-mediated lymphocyte activation was studied in the presence of whole serum from these patients. Changes in lymphocyte proliferation, measured by carboxyfluorescein succinimidyl ester (CFSE) labeling, were detected, and these changes correlated with the level of anti-HLA antibodies. Results Whole serum containing anti-HLA antibodies inhibited lymphocyte proliferation; this effect correlated with the level of antibodies, as measured by LSA. This inhibitory effect was HLA-specific, as shown by adsorption experiments. We also found that relatively high levels of anti-HLA antibodies were necessary to induce changes in an in vitro model of lymphocyte proliferation. Conclusions Our results demonstrate the clinical utility of detecting anti-HLA antibodies by LSA.