Prostate-specific antigen (PSA) is a serine protease expressed at high levels in prostate epithelium, and elevated PSA in serum is a well-established marker of prostate cancer. Recently, the relative proportions of free PSA and PSA complexed to the serine protease inhibitor alpha 1-antichymotrypsin have become important variables in distinguishing between prostate cancer and benign prostatic hyperplasia. Numerous studies have demonstrated the production of PSA in female tissues such as the breast, and low levels of PSA are present in female sera. The objective of this study was to measure and compare the relative proportions of free PSA and PSA complexed to the serine protease inhibitor alpha 1-antichymotrypsin in the serum of women with breast cancer or benign breast disease or women with no known malignancies. PSA was measured with an established immunoassay for total PSA and a novel immunoassay for free PSA, both of which had a detection limit of 0.001 microgram/liter (1 ng/liter). The percentage of breast cancer patients with free PSA as the predominant molecular form (> 50% of total PSA) in serum was five times higher than that of healthy women or women with benign breast disease, and PSA decreased in the serum of breast cancer patients after surgery. The diagnostic use of free PSA for breast cancer is limited at this point, due to the low diagnostic sensitivity (approximately 20%); however, free PSA as the predominant molecular form shows a high diagnostic specificity (approximately 96%) in comparison to women free of breast cancer or with benign breast disease. These results suggest that the clinical applicability of free PSA for breast cancer diagnosis and the biological mechanism behind its increase should be further investigated.