The authors have recently reported that rose bengal is not a vital dye, and stains whenever the cultured cells are not covered by such components as albumin and mucin, and such a tear substitute as carboxycellulose. In this report, using cultured cells as well as normal rabbit corneas, they characterize and correlate the staining differences between rose bengal and fluorescein with the differences in their chemical structures. Fluorescein differs from rose bengal in its lack of intrinsic toxicity, photodynamic action, and ability to be blocked by the above-mentioned substances. Fluorescein staining is increased by rapid stromal diffusion and hence can manifest whenever there is disruption of cell-cell junctions. In contrast, rose bengal staining ensues whenever there is deficiency of preocular tear film protection. These experimental data may help interpret the clinical staining properties of these two dyes and enhance the understanding of the pathogenesis of various ocular surface disorders.