The toxicity of various diepoxy compounds used for biomaterials crosslinking was investigated with a cell culture method and compared with an in vivo method. The neutral red uptake by cells was used to count the number of cells still alive after contact with the diepoxy compounds, because this method was more sensitive in cell counting than the other four methods studied in this work. The amount of neutral red taken up by cells depended strongly on the activity of cells in comparison with other methods; only small amounts of neutral red were taken up when cells were in a low activity state even if they were still alive. The in vitro toxicity of diepoxy compounds evaluated by the neutral red method revealed a good correlation with that found by the in vivo Draize test. The in vitro cytotoxicity to a cell line of L929 was closely related to that of primary culture cells of the normal rabbit cornea epidermal cell. The toxicity of diepoxy compounds was lower as their chain was longer, probably because of the lower chemical reactivity. All the diepoxy compounds investigated in this study exhibited lower cytotoxicity than formaldehyde, glutaraldehyde, and a water-soluble carbodiimide.