75 Human soluble MHC class I molecules (sHLA) appear in serum of healthy individuals at concentrations ranging between 1.0-5.0 μg/ml. These levels are highly elevated during inflammatory processes. The function and role of these molecules has remained, however, unclear. The aim of this study was to elucidate their role in modulating T cell function. Alloreactive T cell lines and clones were generated in vitro by co-cultivating EBV-transformed stimulator cells with peripheral blood lymphocytes of healthy individuals. CD8+ cytotoxic T cells (CTL) against HLA-A2 and -B7 were generated, respectively. sHLA were purified by affinity chromatography and analzed by SDS-PAGE and Western Blotting. Pre-incubation of CTL with sHLA abrogated CTL cytotoxicity allospecifically in a concentration and time-dependent fashion. Prolonged incubation of CTL with sHLA induced apoptosis that led to cell death within 6h. Further analysis of the CTL showed that TcR and CD8 expression were not affected by sHLA, but rather CD95L was strongly upregulated. An anti-CD95L antibody blocked sHLA-induced apoptosis suggesting that CD95/CD95L interaction was the major mechanism for the observed apoptosis. Co-stimulation by CD28 ligation rescued T cells from death. Immobilized sHLA triggered CTL degranulation of perforin and granzyme vesicles. This was shown to be independent of the CD95/CD95L pathway. Intact sHLA (45 kD) and recombinant truncated sHLA (36 kD) were prepared to test whether molecular size affects apoptosis-induction. Only the intact sHLA strongly induced apoptosis in the T cells. Thus, sHLA is a novel tool for elimination of activated alloreactive T cells and may be important for tolerance induction. Further, these data underscore that sHLA are sufficient binding cues for T cell modulation.