Current methods are inadequate for the diagnosis of early chronic allograft rejection. The goal of this study was to determine whether ligation of anti‐HLA antibodies to endothelial cells is associated with a distinctive cytokine secretory pattern. Human iliac artery endothelial cells (HIAEC) cultured in vitro were incubated with w6/32, an anti‐HLA class I mAb. Culture supernatants collected daily for up to 4 days were tested for secretion of 13 cytokines using a multiplexed fluorescent microsphere immunoassay. Culture of HIAEC with medium containing mAb w6/32 supported the growth of HIAEC during the 4‐day study period. Levels of the pro‐inflammatory cytokines IL‐1β, IL‐6, IL‐8, and TNF‐α became significantly increased in supernatants of HIAEC incubated with the mAb w6/32. We conclude that ligation of anti‐HLA class I antibodies to HLA class I antigens in endothelial cells initiates an acute inflammatory process and detecting an inflammatory cytokine secretory pattern might be useful to diagnose sub‐clinical chronic allograft rejection. J. Clin. Lab. Anal. 23:157–160, 2009. © 2009 Wiley‐Liss, Inc.