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To evaluate the effects of trypan blue on cell viability and gene expression in human retinal pigment epithelial (RPE) cells.Three concentrations (0.06 mg/ml, 0.6 mg/ml, and 4 mg/ml) of trypan blue were applied to human ARPE19 cells for 1 minute. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RPE cells were sampled daily for 6 consecutive days to assess the effects of trypan blue on cell viability. The effects of trypan blue on the expression of apoptosis related and cell cycle arrest gene expressions including c-fos, c-jun, p53, and p21 were performed using reverse transcription-polymerase chain reaction and immunostaining.The MTT assay showed a concentration dependent suppression effect of trypan blue on cell viability, with higher reduction in the 0.6 mg/ml and 4 mg/ml trypan blue treated groups. No significant change in the expression of c-fos and c-jun was found with all three concentrations of trypan blue. An increase in p53 expression was found in the 4 mg/ml trypan blue treated group at 10–30 minutes after trypan blue application. Immunostaining showed a mild, albeit insignificant, increase of p53 expression in the RPE cells. No significant increase in p21 expression was observed in the 0.06 mg/ml trypan blue treated group but there were significant increases in p21 expression in both the 0.6 mg/ml (p = 0.032) and the 4 mg/ml (p = 0.025) treated groups.Trypan blue may lead to toxicity on cultured RPE cells as indicated by the reduction in cell viability and changes in the expression of apoptosis related and cell cycle arrest genes at higher concentrations. The application of 0.06 mg/ml trypan blue for 1 minute appeared to have no significant effect on cultured RPE.