PURPOSE: To determine whether hypothermia of 8°C can protect cultured human retinal pigment epithelial (ARPE-19) cells and rat retinal ganglion cells (RGC-5) against trypan blue (TB) toxicity. DESIGN: Laboratory investigation. METHODS: ARPE-19 cells and RGC-5 were exposed to balanced salt solution as controls, and 0.05% and 0.5% TB at 37°C, and at 8°C for one minute. The percentage of surviving cells was determined by the resazurin test. RESULTS: TB induced a statistically significant decrease in the percentage of ARPE-19 cells surviving at 0.5% TB at 37°C (P < .01). Conversely, TB induced a statistically significant decrease in the percentage of RGC-5 surviving at all conditions except for 0.05% TB at 8°C (0.05% 37°C; P < .05, 0.5% 37°C and 8°C; P < .01). CONCLUSIONS: These results indicate that reducing the temperature to 8°C has a protective effect against the TB toxicity for ARPE-19 cells and RGC-5 in culture.