The distribution of annexin A5, a cytosolic protein related to gonadotropin releasing hormone action, was examined in the mammary gland of rats by immunohistochemistry with special reference to changes by lactation. Annexin A5 was observed in the interstitial tissues but not alveolar cells in virgin rats, while mammary epithelial cells became positive for annexin A5 in pregnant rats. The intensity of annexin A5 was decreased in lactating rats and dramatically increased after weaning, especially on the nucleus. When pups were removed from their dam at mid-lactation (day 10), annexin A5 was also increased on day 12. Apoptotic epithelial cells detected by terminal deoxynucleotidyl transferase nick end labeling were simultaneously increased. Annexin A5 mRNA expression of mammary tissues was increased after pup removal. These results are the first to demonstrate the distribution of annexin A5 in the mammary glands of lactating rats, and the enhanced expression in mammary epithelial cells after lactation suggests its involvement in mammary epithelial cell involution.