The vascular endothelium of transplanted organs represents an important target for allograft-directed immune responses. Although HLA antigens expressed on graft endothelial cells (EC) can become targets of the host immune response, the role of other, non-HLA-encoded EC antigens has been proposed but is still unclear. The aim of this study was to investigate the presence of and to characterize anti-EC antibodies (AECA) in 57 kidney transplant recipients according to their HLA-immunization status. Flow cytometry in pretransplant sera was used to detect AECA reactive with surface antigens on ABO and HLA-typed primary cultures of arterial ECs, stimulated or not with tumor necrosis factor-alpha (TNFalpha) or interferon-gamma (IFNgamma). FACS analysis revealed the presence of AECA in 47% of HLA-sensitized (PRA = 10%: mostly IgG) vs. 16.0% in nonsensitized patients (PRA < 10%) (p < 0.02). No significant correlation was found between the presence of AECA and acute rejection occurrence and graft outcome. Non-HLA reactive AECA are directed against TNFalpha- and IFNgamma-inducible membrane molecule(s), and react with two predominant antigens of approximately 35 kDa and approximately 50 kDa expressed on ECs but not on B cells. Binding of AECA decreases in vitro EC viability by 50-60% by promoting EC apoptosis, as demonstrated by DNA fragmentation assays.