Traditional treatments of parasitic and bacterial diseases are based on chemotherapeutic compounds such as chloramine-T and CuSO4. Although many compounds are used in fish treatments, their undesirable effects are not known. In this study, glucose 6-phosphate dehydrogenase (G6PD) was purified from rainbow trout (Oncorhynchus mykiss) erythrocytes by hemolysate preparation, ammonium sulfate precipitation and 2', 5'-ADP Sepharose 4B affinity gel chromatography in a single day. The enzyme, with a specific activity of 14.51 EU/mg protein, was purified 906.8-fold with a yield of 70.38%. To check the purity of the enzyme, SDS polyacrylamide gel electrophoresis was performed, which showed a single band. The effects of chloramine-T and copper sulfate (CuSO4) on the G6PD were investigated in vitro. Chloramine-T and CuSO4 had inhibitory effects on the enzyme. I-50 values of the chemotherapeutic compounds were determined by plotting percent activity and K-i values, and types of inhibition were determined for each compound by means of Lineweaver-Burk graphs. In vivo studies showed that G6PD in rainbow trout erythrocytes was significantly inhibited by CuSO4 in one hour but not inhibited by chloramine-T.