Mast cells in the thyroid gland of different mammalian species were compared with respect to endogenous 5–hydroxytryptamine (5– HT) content, ability to store 5–HT synthesized from its administered exogenous precursor, 5– hydroxytryptophan (5–HTP), ability to take up and store exogenous 5–HT, and the ultrastructure of the specific granules. Mast cells in the thyroid glands of mice and rats showed formaldehydeinduced fluorescence attributable to S–HT. These cells were also labeled radioautographically after injection of tritiated S–HTP or 5–HT in mice. The specific granules in mice and rats had a homogeneous, electron—dense inner matrix when viewed in the electron microscope. In contrast, mast cells of bats and dogs showed no formaldehyde—induced fluorescence attributable to 5–HT. These cells, in bats, did not become labeled radioautographically after injection of either tritiated 5–HTP or 5–HT. The specific granules of thyroid mast cells in bats and dogs had a heterogeneous, lamellar inner matrix when viewed in the electron microscope. Thus, mast cells in thyroid glands of different mammalian species are fundamentally different with respect to 5–HT or monoamine mechanisms, and this difference appears to be reflected in the ultrastructure of the specific granules. It is suggested that the involvement of the release of a monoamine, such as 5–HT, from mast cells in the action of thyrotropin is plausible only in mice and rats.(Endocrinology92: 160, 1973)