Cancer invasion and metastasis require action of tumor-associated proteases, which degrade the extracellular matrix. It has been reported that calpain, a calcium-activated neutral protease and a thiol protease regulated by Ca²+;, proteolyzes estrogen recepor (ER) and that calpain may play an important role in the regulation of ER function. In the present study, the activities of calpain were measured in human normal breast tissues and breast cancer tissues stratified by estrogen receptor levels. There were no correlations between calpain activity and tumor size or lymph node involvement. Activities of calpain were significantly higher in breast cancer tissues compared with those of normal breast tissues, and were higher in the ER-positive tumors than in ER-negative ones. These results indicate that calpain is related to mammary malignant transformation and is involved in the regulation of the ER function in breast cancer tissues.