Tumor heterogeneity may adversely affect the flow cytometric measurement of S-phase fraction (SPF) in breast cancer specimens, and 1&20% of breast cancer specimens are not evaluable by flow cytometry due to technical factors such as debris, high coefficients of variation, poor specimen quality, or small sample size. Therefore, we performed this study on 207 specimens of breast cancer in order to determine if the apoptotic rate (AR) could serve as a useful adjunct to flow cytometric SPF measurements in breast cancers. The average AR in each specimen was determined by microscopic examination of tumor tissue that was specifically stained for apoptotic bodies by a commercially available TUNEL (Tdt-mediated dUTP digoxigenin nick end labelling) assay kit. The mean AR (4.5 f 3.0, n = 37) in the high SPF (>lo%) group was significantly (P < 0.01 1 higher than the mean AR (1.3 f 1.2, n = 72) in the low SPF (<6%) group. Although the distributions of AR values in the two groups had substantial overlap, AR values greater than 5.5 per high power field (h.p.f.1 were not observed in the low SPF cases but were present in 13 out of 37 cases with a high SPF. Simple linear regression analyses relating SPF to the mean AR in 57 DNA diploid cases and 41 DNA aneuploid cases yielded a minimal correlation (rZ = 0.21) between the two parameters only in the DNA aneuploid group. We conclude that an elevated AR has an association with high SPF in breast cancers, but the association is too weak to permit the general use of AR as a predictor of SPF. Our study also identified a subset of breast cancers with both a high SPF (>lo%) and a high AR (>5.5/h.p.f.) that may warrant further investigation to determine its clinical significance. o 1996 Wiley-Liss, Inc.