Diverse pathogenetic factors may lead to the complex syndrome of early graft dysfunction, an important determinant of later renal graft outcome. That humoral factors could play a prominent role in the development of the syndrome was suggested by the capillary deposition of complement fragment C4d in about 50% of graft biopsies. This study investigates whether the presumed classical activation of complement is derived from preformed antibodies that would possibly react against endothelial HLA-class II molecules. Such antibodies were detectable by flow cytometry using a representative collection of 11 DR-typed lymphoblastoid cell lines (LCL) as targets. Simultaneous discrimination between complement-activating and -nonactivating antibodies was achieved by two-color FACS analysis. Using this method, 44 out of 86 pretransplant serum samples from recipients with early dysfunction showed reactivity against LCL (18 complement-activating, 14 nonactivating, 12 complement-activating non-IgG). Conventional panel-reactivity was observed in 20 sera only (14 also LCL-reactive). Evaluation of corresponding graft biopsies revealed that capillary C4d was associated with LCL (P = 0.018) and panel reactivity (P = 0.015) alone and in combination (P = 0.001; Pearson's chi-square test). Thirteen subsequent graft losses within one year were observed in the LCL-reactive group as compared with seven losses in the nonreactive group (panel-reactive: 7; nonreactive: 13). Thus, measurement of LCL-reactive antibodies in prospective transplant recipients improves the assessment of an individual immunological risk. The results further demonstrate that performed antibodies do not simply reflect the enhanced overall immune reactivity of certain recipients but rather act locally in vivo, thus emphasizing the role of humoral factors in the development of early graft dysfunction.