Hydroxyindole-O-methyltransferase was purified from bovine and chicken pineal glands to apparent homogeneity and their properties were compared. The purified enzymes from both pineal glands differed in electrophoretic mobility and isoelectric point. Sodium dodecyl sulfate gel electrophoresis revealed that hydroxyindole-O-methyltransferase of both bovine and chicken pineals was a dimer consisting of a subunit of molecular weight 39,000. The two enzymes also differed in substrate specificity. Bovine hydroxyindole-O-methyltransferase showed a high specificity toward N-acetylserotonin, whereas chicken enzyme methylated N-acetylserotonin and, to some extent, serotonin and bufotenine. The methylation of the three substrates was probably catalyzed by the same enzyme of chicken pineal, because the ratio of substrate availability did not change throughout the purification steps. Using the purified enzymes, we prepared antibody to both bovine and chicken hydroxyindole-O-methyltransferase. The antibody to bovine enzyme cross-reacted with both avian and mammalian enzymes, whereas the antibody to chicken hydroxyindole-O-methyltransferase reacted with avian enzymes, but far less with mammalian enzymes, indicating an immunochemical difference between avian and mammalian hydroxyindole-O-methyltransferase. The results suggest that the properties of hydroxyindole-O-methyltransferase have changed during the evolutionary development of the pineal glands.