The presence of iodothyronine deiodinases was investigated in the different tissues of blue tilapia (Oreochromis aureus), and their biochemical properties were compared with those of mammalian deiodinases. High-Km rT3 outer ring deiodination (ORD) was observed in tilapia kidney, low-Km T4 ORD in liver, and low-Km T3 inner ring deiodination (IRD) in brain and gill. The rT3 ORD activity in tilapia kidney has a very similar substrate specificity as rat liver type I iodothyronine deiodinase but is much less sensitive to inhibition by propylthiouracil, iodoacetic acid, and aurothioglucose. Tilapia liver T4 ORD activity and tilapia brain and gill T3 IRD activities show very similar substrate specificities as well as similar inhibitor sensitivities as rat type II and type III iodothyronine deiodinase, respectively. The optimal pH of the tilapian enzymes is 6-7, and the optimal incubation temperature is approximately 37 C. All tilapia deiodinases are stimulated by dithiothreitol, but the optimal DTT concentrations are generally lower than those required by the corresponding rat enzymes. The apparent Km values of the various tilapia deiodinases for their preferred substrate are in the same range as for the corresponding rat enzymes. Based on these findings, we conclude that fish deiodinases are more similar to mammalian deiodinases than generally accepted.