The aetiology of twin-twin transfusion syndrome (TTTS) is unclear. We investigated the hypothesis that monochorionic (MC) pregnancies with TTTS are associated with differences in the timing and symmetry of twinning compared to MC twin pregnancies without TTTS. DNA was extracted from the umbilical cord vessels of 26 female MC twins, 14 with and 12 without TTTS on serial antenatal ultrasound. X-inactivation patterns were determined by DNA digestion with Hhal and Hpall followed by polymerase chain reaction for a polymorphic trinucleotide repeat in the androgen receptor gene. Products were quantified by densitometry and results compared to those in peripheral blood samples of adult female controls. The median degree of non-random inactivation was similar in MC twins with TTTS, in MC twins without TTTS, and in adult controls. The percentage of individuals with skewed (> or =30/70%) inactivation patterns was no different in MC twins with TTTS compared to those without TTTS, and was similar to adult controls using either enzyme technique. In conclusion we found no difference in the degree or frequency of non-random X-inactivation patterns in TTTS. X-inactivation patterns do not appear to be a useful tool for studying the symmetry of inner cell mass splitting in monochorionic twins.