The in vitro and in vivo effects of melatonin on rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase (GOD) were investigated. G6PD was purified with a specific activity of 16.7 EU/mg protein and 1,852-folds in a yield of 60.6% by using ammonium sulphate precipitation and 2', 5'-ADP Sepharose 4B affinity gel. In vitro studies showed that G6PD enzyme activity increased up to 0.22 mM melatonin concentration and was inhibited at higher levels. In vivo studies showed that though initial G6PD activity was 8.33 +/- 1.13 EU g(-1) Hb, it was inhibited (P < 0.05) 3h after injection of 10 mg kg(-1) melatonin. It is recommended that intramuscular dose of melatonin for rainbow trout should be less than 0.22 mM.