Degradation of the myometrium contributes to the development of adenomyosis and disruption of the actin cytoskeleton occurs in the myometrium adjacent to the invasive endometrium. A subtractive cDNA library that contained genes up-regulated in the uterus with adenomyosis induced by pituitary grafting was constructed in mice. Thymosin beta 4 (Tb4), an actin sequestering protein, was contained in the library and was confirmed to be abundant in the adenomyotic uteri. Tb4-immunoreactivity was shown in the myometrium, with a stronger signal being observed in the area closer to the tip of endometrial tissue that is invading the myometrium. Up-regulation of Tb4 gene expression may be responsible for the disintegration of the musculature during the development of adenomyosis.