Human corneal endothelial cell monolayer preparations were incubated with fresh nonadherent peripheral blood lymphocytes. Vital and supravital staining and direct immunofluorescent techniques were used to analyze lymphocyte binding, endothelial cell viability, and the phenotypes of the lymphocyte populations binding to endothelial cells. Spontaneous lymphocyte-endothelial cell conjugate formation appeared maximal after six hours of incubation (86 lymphocytes per 1,000 endothelial cells). Cytotoxicity assays showed decreased endothelial viability after exposure to the lymphocytes when compared to controls incubated in media alone. The binding-lymphocyte population was a large granular nonadherent lymphocyte lacking surface immunoglobulin and stained positively for Leu-11 antigen, a surface marker expressed by natural killer cells. These data demonstrate that nonsensitized peripheral blood lymphocytes recognize and kill corneal endothelial cells in vitro. Thus, the lymphocyte subpopulations responsible for the destruction of corneal endothelial cells have functional and phenotype characteristics similar to natural killer cells and may be important participants in corneal graft injury.