To date, immunosuppressive therapy for allograft rejection is based on a generalized inhibition of the recipient's T cells, rendering the individual less resistant to infections and malignancies. In order to change this therapeutic approach towards the induction of specific transplant tolerance, it is essential to identify the cells and molecular pathways involved in direct allorecognition. An in vitro model with interferon-gamma (IFN-gamma)-stimulated human lung microvascular endothelial cells (HMVEC-L) as targets and allogenic T cells as responders was used to identify donor cells for recipient cellular immunorecognition. HMVEC-L activated purified allogenic T cells in cocultures. This activation was partly mediated by lymphocyte function antigen-3 (LFA-3), but not CD86, as shown by monoclonal antibody (mAb) inhibition. This finding was supported by the expression of LFA-3 antigen, but not CD86, on IFN-gamma-stimulated HMVEC-L. Surprisingly, even in the absence of T-cell proliferation, T cells were capable of enhancing LFA-3 antigen, but not CD86 expression on HMVEC-L. In conclusion, HMVEC-L are capable of direct allostimulation of human T cells, partly through an LFA-3-dependent costimulatory pathway. Since ICAM-1 expression on HMVEC is greatly enhanced by IFN-gamma and T cell coculturing, this molecule may serve as an additional costimulator. A reciprocal HMVEC-L stimulation by allogenic T-cells occurs, even without T-cell proliferation, possibly representing a preproliferative phase. Since this study included a single target as well as responder cell donor, further studies with multiple donors are needed to evaluate possible variations.