Objective: To report the midterm outcomes of autologous limbal stem cell transplantation cultivated on amniotic membrane (AM) with or without subsequent penetrating keratoplasty (PKP) in patients with total unilateral limbal stem cell deficiency (LSCD). Materials and Methods: Eight eyes of 8 consecutive patients with unilateral total LSCD underwent autologous limbal stem cell transplantation cultivated on AM. Four eyes underwent subsequent optical PKP. Main outcome measures were corneal vascularization and transparency. Results: The patients were followed for 34.0 ± 13.5 months (6 to 48). Seven cases had a stable corneal epithelium with marked decrease in opacification and vascularization. Progressive sectorial conjunctivalization was evident in all cases with subsequent PKP at the last follow-up. Primary failure was observed in one case due to exposure. Conclusion: Transplantation of autologous stem cells cultivated on AM with or without subsequent PKP seems to be an effective way for visual rehabilitation in total LSCD. More work with more cases and longer follow up are needed to optimize this procedure in order to provide and maintain an adequate supply of limbal stem cells in these patients.