We conducted a prospective ancillary study to the study of tamoxifen and raloxifene (STAR) trial involving 4 institutions to determine: (1) our ability to collect nipple aspirate fluid (NAF) in postmenopausal women taking medication to decrease breast proliferation and (2) NAF biomarkers associated with breast cancer. NAF was collected before and 6 months after starting treatment. Three biomarkers, cytology, fluid volume, and prostate-specific antigen (PSA), were analyzed. NAF was collected from 26 of 33 (79%) enrolled subjects at baseline. We were able to collect fluid in 84% of the subjects who produced NAF at baseline and returned for second aspiration after 6 months of treatment. In these women, cytology was unchanged in 85%, improved in 11%, and worsened in 4% of breasts. Median PSA increased from 37.5 to 112 ng/L after treatment. NAF volume did not significantly change. In conclusion, after treatment with tamoxifen or raloxifene, changes in both NAF cytology and PSA were generally favorable, consistent with their expected antiproliferative effective effect on the breast. Multiinstitutional clinical trials in postmenopausal women using NAF biomarkers as the primary endpoints are feasible.