The occurrence and release of thyrotrophin-releasing hormone (TRH) and 5-hydroxytryptamine (5-HT) from amphibian skin have been described by previous investigators. In the present study, the precise location and site of release of TRH and 5-HT from the skin of Rana pipiens and Xenopus laevis have been examined using a combination of procedures including immunohistochemistry, HPLC, and radioimmunoassay. The results indicate that TRH is located specifically within the dermal glands of these species, and that both TRH and 5-HT are discharged from these glands following adrenergic stimulation. The origin and functional significance of these substances in amphibian skin granular glands are discussed.