We have used bivariate flow karyotype analysis to quantify aberrant X chromosome size in 11 XX males. With one exception, the patients could be grouped into those with an X homologue difference greater than normal (Group A, n = 3) and into those whose X homologue difference could not be distinguished from female controls (Group B, n = 7). The range of sizes of the aberrant X chromosome in Ysequence positive patients agrees with the variable nature of the X-Y interchange in these individuals as determined by the use of Y-specific DNA probes and Southern blotting analysis. In one patient it was possible to sort separately the normal and the X-Y interchanged homologues for dot blot analysis. The presence of Y sequences and an increased dose of the zinc finger gene, ZFY, were detected in the X-Y interchanged homologue. In preliminary studies of 5 male and 6 female controls, it was noted that a consistent difference between the two X homologues in females was found which could not be totally explained by errors of the fitting procedure. We suggest that this difference could be due to X inactivation and that the two X homologues in females might be distinguishable.Key terms: X homologue difference, ZFY, variable X-Y interchange XX males are individuals who are phenotypically male (i.e., develop testes) but possess an apparently normal female karyotype without a Y chromosome (8,28). The hypothesis that abnormal X-Y interchange during paternal meiosis results in the transfer of male determinants from the Y to the X chromosomes (14) has been supported, in a proportion of cases, by cytogenetic evidence (6,10,19,20,22,32) and the use of cloned Y-linked sequences (2,5,7,9,12,16,21,(25)(26)(27).Studies using large numbers of Y-specific sequences have shown variable transfer of Y chromosome material in XX males (2). However, quantitative assessment of X chromosome DNA content by using univariate flow karyotype analysis (15) reveals only two groups of XX males. The first group (Group A) demonstrates one X chromosome increased in DNA content while the second group (Group B) is indistinguishable from normal females. In addition, Group A XX males tended to be positive for many more cloned Y-specific