DNA content, proliferative activity (Ki-67 immuno-staining and S-phase fraction by flow cytometry), and neu-oncogene overexpression were studied in 135 patients with invasive breast carcinoma. Image analysis and flow cytometry of fresh tumors showed good correlation between the two methods and yielded 39% diploid tumors and 61% aneuploid tumors. Aneuploidy, including tetraploidy, was significantly related to the loss of estrogen (p = 0.0002) and progesterone (p = 0.03) receptors, high histologic (p = 0.014) and nuclear (p less than 0.0001) grades, and mitotic rate (p = 0.0001). Immunohistochemical evaluation of proliferation by staining with Ki-67 monoclonal antibody and of neu-oncogene protein overexpression was performed in fresh frozen tissue from 83 tumors. The Ki-67 score, quantitated by the CAS-200 image analyzer, correlated only moderately with S-phase fraction obtained by flow cytometry by linear regression analysis (r = 0.39, p less than 0.001). However, both of these proliferation markers correlated strongly with the mitotic rate (p less than 0.0001). Aneuploid and tetraploid tumors demonstrated higher Ki-67 scores and S-phase fractions than diploid tumors. Neu-oncogene protein overexpression was seen in 24 tumors (29%) overall and was much higher in aneuploid tumors (38%) and tetraploid tumors (50%) than in diploid tumors (7%). However, the concentration of neu-oncogene protein positive tumors in the tetraploid region reported by others was not observed. Neu-oncogene protein overexpression was also associated with higher Ki-67 scores (p = 0.016) and S-phase fractions (p = 0.037).(ABSTRACT TRUNCATED AT 250 WORDS)