Sex determination in mammals is controlled by the Y chromosome located SRY gene. Despite recent advances towards understanding the mechanisms that regulate sex determination in mammals, the expression profile of the SRY protein in human tissues is unknown. To localize the SRY protein and determine its cellular distribution, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One antibody, LSRY1.1, recognizes a SRY-specific epitope and was used to localize the protein in different cells and tissues. The mAb recognizes a protein of 27 kDa in total lysates of HeLa SRYB3 cells. Immunocytochemical staining showed a nuclear localization of the protein. Immunohistochemical studies performed on gonadal tissue of a fetus, a one month-old boy and an adult man, demonstrated the presence of SRY protein in the nucleus of Sertoli and germ cells. In addition two 46,XX SRY(+) males had the SRY protein in their gonadal tissues. All other samples were negative, including all female tissue studied and the testis of a 46,XX SRY(-) male. The presence of SRY protein in fetal and adult gonadal tissues including germ cells suggests that SRY may have other male-specific functions in addition to sex determinism.