The effects of melatonin hormone on rainbow trout (Oncorhynchus mykiss) erythrocyte carbonic anhydrase enzyme purified by using Sepharose 4B-L tyrosine-sulfanylamide affinity gel chromatography were investigated in vitro and in vivo. Carbonic anhydrase from Oncorhynchus mykiss erythrocytes was purified with a specific activity of similar to 400 EU/mg protein and similar to 200-fold in a yield of similar to 20. Melatonin did not inhibit carbonic anhydrase enzyme in vitro. Carbonic anhydrase activity of the control group was determined as 1597.7 +/- 429.0 EU/g hemoglobin in vivo. Carbonic anhydrase activities were measured at 1, 3 and 5 h by following melatonin injection. The corresponding mean activities of melatonin were 1406.4 +/- 372.1, 1346.3 +/- 477.4 and 783.8 +/- 494.3 EU/g hemoglobin respectively. Oncorhynchus mykiss erythrocyte carbonic anhydrase was inhibited significantly by melatonin hormone 5 h after injection in vivo (P < 0.05). Data from the present study suggest that excessive usage of this hormone should be avoided in fish reproduction.