Real-time, in vivo, confocal microscopic examination permits visualization of human corneal endothelium cells as bright bodies organized into a densely packed hexagonal arrangement. Quantification of endothelial cell number would be useful in assessing the condition of this cell layer in various disease states. In this study, we sought to use an image analysis method developed in this laboratory that utilizes digital filtering techniques and morphological operations to determine the boundaries of each cell. Images were corrected to establish a uniform luminance level, and then convolved by various matrices until distinct peaks in luminance value were identified. These peaks were used as seed points from which cell boundaries were recursively expanded until they collided with other cell boundaries. This method automatically counts the number of cells and determines the size and position of each cell. The resulting histograms of cell size are readily indicative of changes in cellular density, cell loss, and deviation from uniform arrangement. The numbers of cells counted by this method are consistently within 3% of the numbers counted manually. Results relating cell counts obtained by manual and computerized methods are as follows: 200/184; 276/262; 87/87; 234/232; 236/232; 299/297; 145/147; 119/122; 237/243; 119/119; 245/253; 189/193. Thus, confocal microscopy coupled with these image analysis and statistical procedures provides an accurate quantitative approach to monitoring the endothelium under normal, pathological, and experimental conditions, such as those following surgery and trauma or in the evaluation of the efficacy of topical therapeutic agents.