To examine the density and distribution of Langerhans cells (LCs) in the corneal epithelium of healthy volunteers. Two hundred eyes of 112 healthy volunteers (age 21-81 years) without history of ocular inflammation or surgery were examined in vivo with the combination of the Heidelberg Retina Tomograph II and the Rostock Cornea Module. For statistical analysis data of one eye per volunteer were used, with random selection of one eye in those volunteers in whom both eyes were studied. As studied by in vivo confocal microscopy, 31.3% of all volunteers presented with LCs (24 volunteers with both eyes studied and 11 volunteers with only one eye studied). In 30 of these 35 volunteers, LCs were found in both the central and peripheral corneal epithelium. More than 50% of male volunteers with LCs were younger than 30 years; in contrast, almost two thirds of females with LCs were above 50 years in age. The density of LCs in the periphery of the cornea (98+/-8 cells/mm2; range 0-208 cells/mm2) was significantly (p<0.001) greater than in the central part (34+/-3 cells/mm2; range 0-64 cells/mm2). LCs were located at depths of 35-60 microm, with different frequency. While LCs were sparse at the level of deep intermedial cells (5.7% of the volunteers), 11.4% of the volunteers presented with LCs within the level of basal epithelial cells and most of the eyes (82.9%) had LCs at the level of basal epithelial cells and subbasal nerve plexus. Moreover, LCs presented as either large cells bearing long processes or smaller cells lacking cell dendrites, presumably indicating mature and immature phenotype, respectively. The Heidelberg Retina Tomograph II in combination with the Rostock Cornea Module enables in vivo assessment of density and distribution of LCs in the corneal epithelium, providing insight into human eye immunology. These data may now provide a suitable basis for further investigations in ocular pathology.