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Purpose To assess variability in the coefficient of variation (COV) in cell area estimates when using different numbers of cells for endothelial morphometry. Methods Using non-contact specular microscopy images of the corneal endothelium, 4 sets of 20 cases were selected that included 200 cells and had overall (global) COV values of less than 30 (group 1), 31-40 (group 2), 41-50 (group 3) and over 50% (group 4). Subjects could be normal, or had ophthalmic disease (such as diabetes), a history of rigid or soft contact lens wear or were assessed after cataract surgery. A step-wise analysis was undertaken, 20 cells at a time, of the variability in cell area estimates when using different numbers of cells for the calculations. Results Variability in the average cell area values was higher if only 20-60 cells were used in the calculations and then tended to decrease. The standard deviation values on these average cell area values and the calculated COV showed the same overall trends and were more than twice as large for endothelia with marked polymegethism. Using more than 100 cells/image in markedly polymegethous endothelia only increased the variability in the calculations. Conclusions These analyses indicate that substantial region variability in cell area values can be expected in polymegethous endothelia. The analysis further confirm that using only small numbers of cells (e.g. less than 50/image) in such cases is likely to yield far less reliable estimates of COV.