Metaphase chromosomes derived from leukocyte cultures of peripheral blood were examined in 15 fertile, female, heterosexual bovine twins. Of the 15 animals, 14 contained only XX cells in cultured leukocytes while the remaining female was chimeric containing 26% XY cells. The fertile, female, bovine, heterosexual twin demonstrating XX/XY sex chromosome chimerism in cultured leukocytes supplies evidence that chorionic fusion and vascular anastomosis may occur between male and female cattle twins without production of a freemartin. Although one fertile, female, bovine, heterosexual twin expressed XX/XY chimerism, we believe the leukocyte culture technique is a valuable tool for early diagnosis of the freemartin condition. In comparison with other diagnostic aids including clinical observations, erythrocyte typing, and homograft tolerance, chromosome analysis of leukocytes is equally efficient, less expensive, and more practical for routine early diagnosis of the bovine freemartin. Blood can be sampled soon after birth for karyotyping. From leukocyte culture technique, in 3 days after initiation of the cultures the reproductive capacity of the female twin can be decided.