Anti-H-Y antiserum, raised in highly inbred rats, is absorbed by gonadal cells of various species of fish. This cross-reactivity proved to be restricted to the male sex in the cyprinodont species Lebistes and Xiphophorus, known to have the XX/XY mechanism of sex determination. In members of the more primitive fish orders Isospondyli and Ostariophysi, cross-reactivity was shown to occur as well, but the amount of antiserum absorbed was very similar in both sexes. An antigen cross-reacting with mammalian anti-H-Y antiserum is assumed to exist in fish similar to that found in higher vertebrates. If this is true, this antigen may have been shared originally by both sexes. However, during evolution, its expression has become restricted to the heterogametic sex.