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To compare endothelial cell analysis by the center and flex-center methods in corneas with guttae of differing severity and to determine the minimum countable cell number for using only the flex-center method.Forty-seven eyes with corneal guttae and 50 age-matched normal controls were enrolled in the study. Three images were captured in each central cornea with the noncontact specular microscope. Endothelial cell density (ECD), coefficient of variation (CV), and percentage of hexagonal cells (HEX) were analyzed by trained graders, using both center and flex-center methods.Consistent ECD and HEX values were obtained in normal eyes by both methods (P > 0.05). In corneas with guttae, ECD values obtained by the center method were 2.4% higher than those obtained with the flex-center method (P < 0.001). ECD values derived by both methods disagreed only when <30 cells were identified or <20 cells were analyzed. CV values obtained by the center method were 17.1% (P < 0.001) lower than those obtained by the flex-center method. HEX values obtained with both methods (P > 0.05) agreed. Regardless of guttae density, the ECD, CV, and HEX values of 3 images of each eye were in agreement (P > 0.05).In corneas with guttae, both center and flex-center methods can reliably determine ECD. Although current practice recommends the center method when at least 100 cells can be counted, our study suggests that the center method can provide a reliable ECD value when there are ≥30 contiguous countable cells in a central cornea endothelial image. The flex-center method is recommended when <30 contiguous cells are identified.