Fine-needle aspiration (FNA) is a sensitive and cost-effective method for evaluating breast lesions. However, the diagnosis of early premalignant lesions is less reliable by FNA because of a lack of distinctive cytological features. Accurately defining the risk of such lesions at the individual level may have significant impact in breast cancer prevention and management. The main objective of this preliminary study was to develop a method to study multiple biomarkers on archival FNA slides using quantitative fluorescence image analysis (QFIA). Biomarkers p53, G-actin, and DNA content were labeled with an immunofluorescence technique and measured by QFIA simultaneously on a single cell basis. QFIA allows the labeling and measurement procedures to be carried out in situ, without the need to remove cells from the slide while preserving the morphology of the cells. FNA slides from 72 incident patients were obtained for this study. Fifty-six cases had an adequate number of cells for the actual analysis (25 benign breast lesions, 14 proliferative breast diseases with nuclear atypia, and 17 malignant lesions). The DNA content (> or = 5c) and G-actin (average gray mean, > 90) were positive in 81% and 88% of malignant lesions, respectively. These were significantly higher than the corresponding positive rates in benign lesions (7% and 15%, respectively; P <0.01 for both). None of the benign cases were positive for G-actin and DNA simultaneously, and none of the malignant cases were negative for G-actin and DNA together. p53 was positive in 33% of malignant lesions and 8% of benign lesions (P >0.05). Our study demonstrates the feasibility of evaluating multiple biomarkers by QFIA on archival FNA-fixed specimens. The G-actin and DNA content assayed by QFIA may be potential intermediate end point markers for breast cancer individual risk assessment.