In amphibian epidermis mucus is thought to constitute the matrix material that links keratin filaments present in cells of the corneous layer. As contrast in mammals, and perhaps in all amniotes, histidine-rich proteins form the matrix material. In order to address the study of matrix molecules in the epidermis of the first tetrapods, the amphibians, an autoradiographic and electrophoretic study has been done after administration of tritiated histidine. Histological analysis of amphibian epidermis shows that histidine is taken up in the upper intermediate and replacement layers beneath the corneous layer. Ultrastructural autoradiographic analysis reveals that electron-dense interkeratin material is labeled after administration of tritiated histidine. Electrophoretic analysis of the epidermis shows labeled proteic bands at 58-61, 50-55, 40-45, and some only weakly labeled at 30 and 24-25 kDa at 4-48 hours after injection of tritiated histidine. Keratin markers show that bands at 40-61 kDa contain keratins. Most histidine is probably converted into other amino acids such as glutamate and glutamine that are incorporated into newly synthetized keratins. However, non-keratin histidine-incorporating proteins within the keratin range could also be formed. The bands at 30 and 24-25 kDa suggest that these putative histidine-rich proteins are not keratins. In fact, their molecular weigh is below the range of that for keratins. In contrast with the mammalian condition, but resembling reports for lizard epidermis, putative histidine-rich proteins in amphibians have no high molecular weight precursor. Although filaggrin is not detectable by immunofluorescence in sections of amphibian epidermis, protein extraction, electrophoresis and immunoblotting are more sensitive. In the epidermis of toad and frog, but only occasionally in that of newt, filaggrin cross-reactive proteic bands are seen at 50-55, 40-45, and sometimes at 25 kDa. This suggests that after extraction and unmasking of reactive sites in the epidermis of more terrestrial amphians (anurans), some HRPs with filaggrin-like cross-reactivity are present. The overlap that exists at 50-55 kDa between filaggrin-positive and AE2-positive keratins, but not that at 40-45 kDa further indicate that non-keratin, filaggrin-like proteins may be present in anuran epidermis. The present study suggests for the first time that very small amounts of histidine-rich proteins are produced among keratin filaments in upper intermediate, replacement and corneous layers of amphibian epidermis. Although the molecular composition of these proteins is unknown, precluding understanding of their relationship to those of mammals and reptiles, these cationic proteins might have originated in conjunction with the formation of a horny layer during the adaptation to land during the Carboniferous and were possibly refined later in the epidermis of amniotes.