We previously demonstrated that nipple aspirate fluid (NAF) can be obtained from virtually all non-Asian women between the ages of 30 and 72. The focus of this report is to (1) determine the association of candidate markers of breast cancer risk in NAF obtained from fresh mastectomy specimens with residual breast carcinoma, and (2) evaluate the association of the markers with breast tumour progression. Nipple aspiration was performed on 97 specimens. Cytology, DNA index (including % hypertetraploid cells), cell cycle parameters (S phase fraction, % cells in G2/M), prostate-specific antigen (PSA), epidermal growth factor (EGF), testosterone, carcinoembryonic antigen (CEA) and prostaglandin D synthase (PGDS) were evaluated in NAF for their association with (1) residual ductal carcinoma in situ (DCIS) or invasive cancer, and (2) pathologic tumour size. NAF was obtained from 99% (96/97) of specimens. Atypical and malignant NAF cytology were significantly associated with residual DCIS or invasive cancer (P = 0.001) and with larger tumours (P = 0.004). One hundred per cent and 88% of subjects with malignant and atypical NAF cytology, respectively, had residual carcinoma. The percentage of cells in G2/M and DNA index were associated both with risk of residual carcinoma (P = 0.01 for each) and larger tumour size (DNA index, P = 0.03; G2/M, P = 0.05), although neither biomarker improved the ability of NAF cytology, to predict residual breast cancer. Higher DNA index was associated with atypical cytology (P = 0.0001). In summary, atypical and malignant NAF cytology are associated with larger tumour size, and are highly predictive of residual carcinoma after needle or excisional biopsy of the breast.