The influence of ultraviolet (UV) irradiation on creosote toxicity was investigated with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, and two indicator dyes, alamar Blue and 5-carboxyfluorescein diacetate acetoxymethyl ester. These monitor redox potential and membrane integrity, respectively. After solubilization and chemical analysis, creosote was presented to cells in the dark to measure cytotoxicity or concurrently with UV irradiation to evaluate photocytotoxicity. Additionally, creosote was photomodified by 2 h of UV irradiation before presentation to cells in the dark or together with UV. Cytotoxicity was detected only at high nominal creosote concentrations, but photocytoxicity occurred at creosote concentrations 35-fold lower. All the aromatic hydrocarbons in creosote appeared to contribute to cytotoxicity, but photocytotoxicity was due only to the fluoranthene, pyrene, anthracene, and benzo[a]anthracene in the mixture. Photomodified creosote was much more cytotoxic than intact creosote and this difference was most pronounced in the alamar Blue assay. Likely, this was due to photomodification products that impaired the mitochondrial electron transport chain. Photomodified creosote was slightly less photocytotoxic than intact creosote. Overall these results indicate that UV irradiation potentially enhances the toxicity of creosote to fish in several different but significant ways.