Although cytomegalovirus (CMV) interferes with major histocompatibility expression in infected cells, both host and donor soluble human leukocyte antigen class I (sHLA-I) are often released into the serum of transplant recipients during CMV infection and may contribute to anti-HLA antibody production and graft rejection. We hypothesized that CMV infection of endothelial cells (EC) induces host T cells to release interferon (IFN)-gamma, which in turn drives the metalloproteinase (MPase)-cleavage pathway of sHLA-I generation in "bystander" uninfected ECs. To test this hypothesis, cultures of peripheral blood mononuclear cells (PBMCs) and either uninfected ECs or CMV-infected ECs (EC/CMV) were established and supernatants were tested in enzyme-linked immunosorbent assay for sHLA-I. Responder PBMC became activated and released sHLA-I via the MPase pathway when stimulated with allogeneic EC/CMV; the sHLA-I release was contact dependent and cytokine independent. In transwell cultures, IFN-gamma released by PBMCs in response to EC/CMV stimulated a release of sHLA-I from uninfected allogeneic ECs across the transwell; this release was also MPase dependent. This implies that CMV infection within the transplanted allograft will not only stimulate the release of self HLA from responding PBMCs, but will also stimulate the release of donor sHLA-I from uninfected bystander ECs, both via the class I MPase-pathway.