46,XX subjects carrying the testis determining SRY gene usually have a completely male phenotype. In this study, five very rare cases of SRY carrying subjects (two XX males and three XX true hermaphrodites) with various degrees of incomplete masculinisation were analysed in order to elucidate the cause of sexual ambiguity despite the presence of the SRY gene. PCR amplification of 20 Y chromosome specific sequences showed the Yp fragment to be much longer in XX males than in true hermaphrodites. FISH analysis combined with RBG banding of metaphase chromosomes of four patients showed that in all three true hermaphrodites and in one XX male the Yp fragment was translocated onto a late replicating inactive X chromosome in over 90% of their blood lymphocytes. However, in a control classical XX male with no ambiguous features, the Yp fragment (significantly shorter than in the XX male with sexual ambiguity and only slightly longer than in XX hermaphrodites) was translocated onto the active X chromosome in over 90% of cells. These studies strongly indicate that inactivation on the X chromosome spreading into a translocated Yp fragment could be the major mechanism causing a sexually ambiguous phenotype in XX (SRY+) subjects.