Hydroxyindole—O—methyl transferase activity (HIOMT) is present in rat pineal, retina and harderian gland. We have studied the biochemical properties of HIOMT activities in homogenates of these tissues by measuring the transfer of a 14C—methyl group from S—adenosylmethionine to N—acetylserotonin. Melatonin synthesis was linear for 30 min; pH optima were 7.5–8.4 (pineal and retina) and 7.3–7.9 (harderian gland). The HIOMT activity in pineal and retina, but not in harderian gland, decayed to 35% of initial value after 5 days of storage at –20 C. Mg++ enhanced the activity of the harderian gland enzyme, but had no effect on pineal or retinal HIOMT; sulfhydryl blocking agents inhibited all three enzymes. N—Acetylserotonin was the best indolic substrate (i.e., highest maximal velocity) for all three. The relative O—methylations of 5–hydroxyindoleacetic acid, 5–hydroxytryptophol and serotonin were similar in all these cases. The Km for S—adenosylmethionine was about 10–55M for the three enzymes and the Km for N—acetylserotonin was also about 10–5M for the HIOMT activity in pineal and retinal homogenates; however, it was more than 100–fold greater (1.2 X 10–3M) for harderian gland HIOMT. These data suggest that the HIOMTs in pineal and retina are closely related enzymes. The markedly greater Km of the harderian enzyme for N—acetylserotonin raises the possibility that its function in vivo involves other, nonindolic, substrates. (Endocrinology91: 247, 1972