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Purpose Diagnosis of graft rejection is based on patient symptoms and on clinical signs detected by slit-lamp biomicroscopy. This study investigated whether laser in vivo confocal microscopy (IVCM) can aid in the diagnosis of corneal graft rejection by detecting cellular corneal changes that take place after transplantation. Design Prospective case-control study. Subjects Thirty-eight eyes of 38 patients with penetrating keratoplasty (15 eyes with corneal graft rejection, 23 eyes without rejection) and 9 age-matched normal controls. Methods Laser IVCM was performed in the corneal grafts centrally. The density of immune cells (IC) was assessed for epithelial, sub-epithelial, stromal, and endothelial layers by 2 masked observers. IC density was compared among different groups and correlated to clinical signs and symptoms of corneal graft rejection. Main Outcome Measurements Outcome measurement was the IC density in the corneal layers and its associations with the presence of clinical signs and symptoms of corneal graft rejection. Results The IC density was significantly different between rejected and non-rejected grafts (P = 0.004) and different from that of normal controls (P = 0.001). Among corneal layers, IC density was significantly higher in rejected grafts than in non-rejected grafts in only the sub-basal (611.54 ± 573.74 vs. 340.61 ± 268.60 cells/mm2, respectively; P = 0.049) and endothelial layers (250.62 ± 267.13 vs. 103.47 ± 81.91 cells/mm2, respectively; P = 0.001). Patients with decreased best corrected visual acuity, Khodadoust line, and anterior chamber cells demonstrated a significant increase in total IC density (P Conclusions Patients with corneal graft rejection demonstrate a significant increase in corneal immune cells, particularly, in the sub-basal and endothelial layers compared to patients with non-rejected grafts and controls. Although symptoms associated with endothelial rejection demonstrate a general increase in IC, pain, irritation, and light sensitivity are associated with increased IC in the sub-basal layer. Assessment of patients with corneal graft rejection by IVCM may serve as an adjunctive tool in the diagnosis and management of corneal graft rejection.