BACKGROUND: The purpose of the study was to map the precise location of four antioxidant enzymes in the normal human conjunctiva. METHODS: Conjunctival tissue (seventeen specimens) from seven patients was processed for light microscopy and immunogold electron microscopy. Antibodies were used to label glutathione peroxidase, glutathione S-transferase (acidic and neutral forms) and copper/zinc superoxide dismutase. RESULTS: All layers of the epithelium exhibited labelling for all four enzymes, although there were differences in labelling intensities and distribution between the enzymes. Wing cells showed more intense labelling for glutathione peroxidase, glutathione S-transferase (neutral form) and copper/zinc superoxide dismutase than did basal and superficial cells. The strongest immunoreactivity was detected for glutathione S-transferase (acidic) and was equally strong in the superficial and wing cells. Wing cells demonstrated more immunoreactivity in the cell nuclei with antibodies against all four enzymes. A high degree of labelling was also observed in the microplicae and actin cortex of the superficial cells. Leukocytes, fibroblasts, endothelial cells and erythrocytes within the stroma exhibited minimal labelling. CONCLUSION: The subtle changes in distribution of antioxidant enzymes in the normal human conjunctival epithelium most probably reflect exposure to free radicals in the tear film and are based on the different proliferative and functional activities of each of the anatomical layers.