BACKGROUND: Free cyanide is a potent toxic agent in the aquatic environment.  Freshwater fish are the most cyanide-sensitive group with high mortality at free cyanide concentrations above 20 μg/L. Exposure to cyanide ions can cause stress, increased mortality and place an appreciable metabolic load on fishes. Rhodanese is a ubiquitous mitochondrial enzyme in both prokaryotes and eukaryotes that detoxifies cyanide (CN-) by converting it to thiocyanate (SCN). OBJECTIVES: The purpose of this investigation was to determine and compare the pattern of tissue distribution of Rhodanese in different tissues of four native Barbus fish including Mesopotamichthys sharpey, Tor grypus, Luciobarbus xanthopterus and Luciobarbus barbulus. METHODS: Fishes (10 from each species) with length of 32.5 ± 6.5 and weight of 440 ± 110   were collected from five major fishing reservoirs of Karoun River including Gotvand, Shushtar, Molasani, Darkhoine and Ahvaz. Rhodanese activity was assayed by the method of Sorbo in the liver, kidney, gill and intestine. The unit of enzyme activity was defined as micromoles thiocyanate formed per minute at 37 °C and pH 9.2 and enzyme activity was expressed as U/mg protein. RESULTS: Rhodanese activity was detected in all tissues studied, albeit in different amounts. Specific activities of Rhodanese (U/mg protein) in different tissues ranged from 0.135 to 0.337 in the liver, 0.113 to 0.262 in the kidney, 0.121 to 0.157 in the gill, and 0.094 to 0.162 in the intestine, respectively. CONCLUSIONS: The highest activity of Rhodanese in all four species was observed in the liver and kidney, followed by the gill and intestine. Our results suggest that Rhodanese may be functional in many physiological activities in these species which needs to be clarified in detailed.