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BACKGROUND The goal of the prospective study was to determine whether 1) image analysis (IA; including DNA index [DI], S-phase fraction, and the presence or absence of aneuploidy or hypertetraploidy [HT]) of fiberoptic ductoscopy (FD) breast specimens was feasible, 2) IA findings from FD specimens predicted histopathologic evidence of disease, and 3) a combination of IA, cytology, and clinical factors provided complementary information in the diagnosis of breast carcinoma. METHODS IA and cytologic evaluation were performed on 106 consecutively collected ductoscopic specimens from 88 subjects. RESULTS IA was successful in 73 (71%) FD specimens. HT (P = 0.03) was related to intraductal visual observations. The proportion of atypical papillomas with aneuploidy was greater (P = 0.05) than in any other class. The HT index was higher in atypical papilloma (P = 0.02) and in breast carcinoma (P = 0.05) than with other diagnoses. The proportion of papilloma cases with HT was greater (P = 0.04) than benign papillomas. Malignant cytology was associated with a higher DI (P = 0.02) and a higher HT index (P = 0.001) than nonmalignant (benign or atypical) cytology. HT (P = 0.002) was less common with cytology containing few epithelial cells, and the HT index was higher with malignant versus nonmalignant FD cytology (P = 0.001). The percentage of cells containing HT was greater in FD specimens that were suspicious for malignancy (P = 0.006) than in those with benign cytology or mild atypia. Considering all samples and combining IA, cytologic, and visual findings in a stepwise linear discriminant analysis optimized the sensitivity (61%) and specificity (90%) of breast carcinoma prediction. Excluding spontaneous nipple discharge (SND) samples and adding epithelial cell quantity in samples improved the model (sensitivity, 85%; specificity, 80%). CONCLUSIONS IA was feasible in FD specimens whenever adequate epithelial cells were present. IA and cytologic findings were associated. Adding IA results to cytology and visual findings and excluding samples with SND improved diagnostic sensitivity. Cancer 2004. © 2004 American Cancer Society.