It has been proposed that the male-specific transplantation antigen H-Y is the trigger for testis formation (Ohno's hypothesis). We have tested this hypothesis by examining the H-Y status of a number of mice with abnormal or aberrant karyotypes relative to their gonad development. This analysis includes the discussion of published results of XX males carrying the Sxr mutation and XO females, and the presentation of unpublished data from XY females carrying the YPOS or YORB chromosome, XY females carrying Thp on chromosome 17, and a series of mice carrying X-Y recombination products derived from the Y* chromosome. The XX Sxr males have testes and are H-Y transplantation antigen positive; XO females have ovaries and are H-Y negative; mice with X-Y reciprocal recombinant chromosomes resulting from an abnormal Y chromosome show concordance between the presence of testes and the presence of the H-Y antigen. All of these findings are in accord with Ohno's hypothesis. In contrast are our findings that three types of C57BL/6J XY females are H-Y positive, two inheriting a Y chromosome from the mouse species Mus domesticus, either YPOS or YORB, and the other a mutation associated with Thp. Genetic analysis of the inheritance of this paradoxical phenotype indicates that normal testis differentiation involves the interaction of two or three genes, and is not simply a function of a Y-linked gene product. We conclude that H-Y is not the sole Y-linked testis determining gene.