The S-phase which assesses tumor proliferation has been considered to be an independent prognostic factor for breast carcinoma. Quantitative analysis of MIB-1 immunoreactivity is a newly recognized method of determining cellular proliferation that offers some advantages over flow cytometry when limited tumor tissue is available. However, it has been controversial whether there is a significant correlation between MIB-1 immunostaining and S-phase in defining proliferation activity in breast cancer. In order to explore the usefulness of MIB-1 as an additional proliferation parameter and a potential prognostic factor for breast cancer, we analyzed 94 cases of invasive ductal carcinoma of the breast by both flow cytometry (for S-phase and DNA ploidy) and quantitative MIB-1 immunohistochemical analysis using formalin-fixed paraffin-embedded tissue. MIB-1 staining was quantitatively analyzed by image analysis and by visual scoring. Forty-six cases were diploid by flow, while the remaining 48 cases were aneuploid tumors. T-test results indicated that S-phase means were significantly greater (p = 0.0001) in aneuploid cases (mean = 18) compared to diploid cases (mean = 7). MIB-1 means were also greater in aneuploid patients, but these differences were only marginally significant (p = 0.05). S-phase was positively correlated with MIB-1 (r = 0.36, p = 0.003 for image analysis and r = 0.34, p = 0.001 for visual scoring). ROC curve analysis indicated that MIB-1 quantitation is a good predictor of high S-phase (i.e., > 10 percent) in aneuploid cases. A MIB-1 cutoff value of 25 percent for image analysis achieved 82 percent specificity and 80 percent sensitivity for aneuploid high S-phase, while a MIB-1 cutoff value of 40 percent for visual scoring was 73 percent specific and 85 percent sensitive. However, in diploid cases, no comparable MIB-1 cutoffs could be achieved for detecting high S-phase. In summary, our study demonstrated that aneuploid breast carcinomas proliferate more aggressively than diploid tumors. Although linear correlation between MIB-1 and S-phase was weak, MIB-1 was considered to be a good predictor of high S-phase in aneuploid breast cancer patients, possibly due to a threshold effect. Image analysis and visual scoring of MIB-1 immunoreactivity appeared to be comparable in analyzing proliferative activity in breast cancer. Thus MIB-1 assessed by visual scoring may be a less expensive alternative to image analysis.