The proliferative capacity of breast carcinomas has prognostic significance as measured by S-phase fraction (SPF), yet the molecular parameters that influence the proliferative capacity of breast carcinomas have not been fully elucidated. Ninety-three cases of invasive ductal breast carcinomas, not otherwise specified, were studied by immunohistochemistry and flow cytometry to determine what correlations exist between nuclear grade (NG), SPF, proliferating cell nuclear antigen and the overexpression of p53, epidermal growth factor receptor (EGFR), and c-erb-B-2 in formalin-fixed tissues by the immunoperoxidase method. NG predicted elevated SPF in 78% of cases and was associated with high proliferating cell nuclear antigen score. The presence of p53 was detectable in 13% of cases, and, in each case, the NG was high (Grade 3), with nine aneuploid tumors and three diploid tumors. The SPFs for all p53-positive cases were markedly elevated, with 77.8% of the cases with SPF > 15%. EGFR was present in 20.4% of all tumors, including 77.8% of tumors positive for p53 and 14% of tumors negative for p53 (P < .001). The mean SPF for p53-positive EGFR-positive tumors was 17.5% versus 21.1% for p53-positive EGFR-negative tumors. The mean SPF for p53-negative tumors was significantly less, regardless of the presence of EGFR. The gene c-erb-B-2 was found in 28% of tumors, all of which were p53 negative. These data clearly show a close relationship between high NG and elevated SPF. As determined by flow cytometry, SPF is more consistent and more reliably related to NG than proliferating cell nuclear antigen.(ABSTRACT TRUNCATED AT 250 WORDS)