The mammalian Y chromosome encodes a testis-determining factor (termed TDF in the human), a master regulator of sex differentiation. Embryos with a Y chromosome develop testes and become males whereas embryos lacking a Y chromosome develop ovaries and become females. Expression of H-Y, a minor histocompatibility antigen, may also be controlled by a gene on the Y chromosome, and it has been proposed that this antigen is the testis-determining factor. We have tested the postulated identity of H-Y and TDF in the human. H-Y typing with T cells was carried out on a series of sex-reversed humans (XX males and XY females), each shown by DNA hybridization to carry part but not all of the Y chromosome. This deletion analysis maps the gene for H-Y to the long arm or centromeric region of the human Y chromosome, far from the TDF locus, which maps to the distal short arm.