Bindings of rose bengal to several proteins were determined by Sephadex G-75 chromatography. Their respective blocking effect against dye uptake was demonstrated in an assay using a rabbit corneal epithelial cell layer. The total binding capacity of nonmucin proteins was measured using fluorometry and Scatchard analysis. The results showed that albumin, lactoferrin, transferrin, and lysozyme could--but serum prealbumin, IgA, carboxymethyl cellulose (CMC), and Sepharose 4B-purified porcine stomach mucin (PSM) could not--bind rose bengal. Lysozyme formed precipitates with rose bengal. Sufficient concentrations of albumin, lactoferrin, transferrin, or lysozyme premixed with rose bengal could block dye uptake by cells, but IgA and serum prealbumin could not. Premixed PSM was not as effective as precoated PSM in blocking dye uptake. The dissociation constant (Kd) was 1.2 x 10(-7) M, 3.6 x 10(-7) M, 3.9 x 10(-7) M, and 1.6 x 10(-6) M for albumin, transferrin, lactoferrin, and lysozyme, respectively. Based on these values, the total maximal binding capacity of nonmucin proteins in normal 7-microliters tears was extrapolated to be 0.249 micrograms rose bengal, which is too small to explain the negative staining of rose bengal on the normal ocular surface. Rose bengal, but not fluorescein, could interact with carbohydrate-containing Sephadex, CMC, and PSM to slow down its elution via Sephadex column chromatography. Therefore, the normal negative staining to rose bengal might be caused by the blocking effect of preocular mucus tear layer, which serves as a diffusion barrier. Rose bengal remains a unique dye for detecting the protective function of the preocular mucus tear.