A probe from a conserved motif of the SRY gene (sex-determining region Y), a prime candidate for the human testis-determinant, was hybridized to DNA from 23 species representing 5 vertebrate classes. Hybridization occurred in species with male or female heterogamety, in species with and without sex chromosomes and in those with temperature sex determination. Sex-specific signals were observed only in mammals. Conservation of sequences homologous with SRY through 400 million years of vertebrate evolution would indicate persistence of function. However, if SRY is the primary sex determinant in mammals, it is not clear that it has a similar function, or even one that is sex-related, in nonmammals.