H-Y antigen was investigated in three amphibian species with different degrees of sex-chromosome differentiation: Bufo bufo, Triturus vulgaris, and Pyxicephalus adspersus. No heteromorphic sex chromosomes were found in B. bufo, but an examination of the progeny of hermaphrodites (Ponse, 1942) indicated that the female of this species was heterogametic (ZW). Sex chromosomes differing only by a very small heterochromatic region at their telomeres were found in the male of T. vulgaris (XY). Pyxicephalus adspersus revealed high differentiated ZW sex chromosomes. The results of the H-Y antigen studies on these three species indicate that H-Y antigen is expressed only in the heterogametic sex, irrespective of differences in morphological differentiation of the sex chromosomes. Therefore, H-Y antigen could be a valuable tool in determining the heterogametic sex, not only in Amphibia but possibly also in other vertebrate species that have either evolved no heteromorphic sex chromosomes or where sex-reversal experiments are not possible.