Three corneoscleral discs (from two donors) underwent subtotal endothelial loss during routine “long-term” organ culture storage. Laboratory studies of these corneas revealed evidence of herpes simplex virus (HSV) infection. The fellow cornea from one of the donors had been issued for transplant to a patient with keratoconus. Deterioration of the graft was noted 5 days after surgery; the disc was removed at 2 months and was shown to be infected with HSV. In an experiment designed to simulate initial “cleansing” of donor globes, 0.1% polyvinylpyrolidone-iodine protected cells from infection with HSV. It was concluded that the detection of HSV in these corneas could not be explained by external contamination of the ocular surface. Furthermore, culture of conjunctival and pharangeal swabs taken from 47 consecutive donors confirmed that HSV is rarely isolated at or around the time of death. Five pairs of donor corneas destined for use in transplantation were selected at random and investigated for the presence of HSV. HSV DNA was detected by polymerase chain reaction (PCR) in tissue from two of the corneal donors. Sequential stepwise sectioning suggested that HSV DNA when present was distributed in discrete foci within the cornea. These observations suggest that HSV infection may be a cause of severe endothelial loss during corneal organ culture and possibly provide an explanation for some “failures” of corneal grafting.