Many clinical studies have been performed investigating corneal endothelial cell loss after penetrating keratoplasty using different preservation methods. Most studies, however, included variable follow-up intervals, neglecting the dynamic cell loss over the course of time. The aim of this study was to perform endothelial cell evaluation 5 years after penetrating keratoplasty using two different storage methods. Fifty-four patients were examined 5.2 (+/-0.5) or 4.9 (+/-0.6) years after surgery. Twenty-four patients had received a cornea stored in a moist chamber, 30 patients a cornea preserved by organ culture. All corneas had remained clear in the follow-up period. The follow-up periods did not differ significantly (P = 0.26). The post-mortem time of moist-chamber-stored tissue was significantly lower (P < 0.001), endothelial cell density significantly higher (P < 0.001) in organ-culture-preserved corneas. Donor age (P = 0.64) and patient age (P = 0.046) did not differ significantly. Average corneal endothelial cell density was 1070 (+/-499) cells/mm2 using moist chamber stored and 1095 (+/-497) cells/mm2 using organ-culture-preserved tissue (P = 0.82). Five years after penetrating keratoplasty, we were not able to find significant differences in corneal endothelial cell density using either moist chamber or organ-culture-preserved corneas. We prefer to use organ culture since there is a lower incidence of primary graft failure, tissue with longer postmortem times can be used, and surgery can be scheduled.