Anuran, such as Brazilian toad Bufo ictericus Spix, 1824, possess a calcified dermal layer called the Eberth-Katschenko (EK) layer located between the stratum spongiosum and the stratum compactum. It has been regarded as a protective barrier against desiccation or involved in storage and mobilization of calcium, or a barrier for the interchange of substances between the animal's internal and external environment. The B. ictericus integument was removed from the ventral and dorsal regions and examined by light microscopy, using histochemical techniques, and also submitted to biochemical calcium analysis. The intermediate layer is strongly basophilic and metachromatic. In the EK layer Alcian blue at different critical electrolytic concentration revealed hyaluronic acid, which coexists with the dermal calcium. We conclude that the EK layer of B. ictericus is a discontinuous layer and is formed of hyaluronic acid and a sulfated GAG different from chondroitin sulfate, heparin and heparan sulfate or keratan sulfate. The amount of cutaneous calcium is independent of region and sex. GAGs may play an important role in dermal hydric balance, protecting the animal against desiccation, and hyaluronic acid is probably able to anchor dermal calcium. Thus, all compounds of the EK layer are essential for integument integrity and functionality.