Melatonin modulates a variety of rhythmic processes in vertebrates, and is synthesized in both the retina and pineal gland. We have shown previously that retinal melatonin is deacetylated generating 5-methoxytryptamine, which is then deaminated by monoamine oxidase, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. This process occurs within the eyes of a variety of vertebrates including the iguanid lizard Anolis carolinensis. To determine whether melatonin deacetylase activity also occurs in the pineal organ or in other parts of the lizard brain, pineals and brains of Anolis carolinensis and Sceloporus jarrovi were cultured in the presence of [3H-methoxy]-melatonin. High-performance liquid chromatography of the resulting culture media and tissues revealed the generation of radiolabeled 5-methoxytryptamine and 5-methoxyindoleacetic acid. These two methoxyindoles were the only radiolabeled metabolites detectable, and together accounted for all melatonin lost. Both the loss of melatonin and the production of melatonin metabolites were inhibited by inclusion of 100 microM eserine, an inhibitor of the melatonin deacetylase. Pargyline, a monoamine oxidase inhibitor, reduced the production of 5-methoxyindoleacetic acid and increased the production of 5-methoxytryptamine relative to control incubations. Similar effects of eserine and pargyline were seen in eyecup, brain and pineal gland, but the specific activity of melatonin deacetylation in cultured pineal glands was much greater than in either brains or eyecups. These results indicate that pineal glands of both Anolis carolinensis and Sceloporus jarrovi can rapidly catabolize melatonin by a mechanism very similar to that in the eye, that the melatonin deacetylation pathway exists elsewhere in the iguanid brain, and also extend our previous observations of ocular melatonin deacetylation to an additional species.