Flow cytometric assessments of DNA ploidy status and the S-phase fraction (SPF) have been shown to yield prognostic information in breast cancer. The aim of the present investigation was to elucidate the reproducibility of results with regard to tumor DNA heterogeneity, and to ascertain whether the prognostic value of DNA measurements might be enhanced by analyzing two pieces of a tumor instead of one. Agreement with regard to ploidy status (diploid versus non-diploid) was obtained in 90% of cases (71/79) when two adjacent sections of the tumor were investigated, and in 77% of cases (10/13) when four biopsies from different quadrants of the tumor specimen were investigated. The corresponding figures for agreement in SPF (divided into three categories, less than 7.0%, 7.0-11.9%, and greater than or equal to 12%) were 75% (59/79; 2-sample series) and 55% (7/13; 4-biopsy series). The main reason for variance in ploidy results was the difficulties in distinguishing near diploid cell populations. Discrepancies in SPF categories could be explained by minor fluctuations in SPF values near the cut-off levels, or by variance in ploidy status, the fraction of non-diploid nuclei, and background noise due to cell debris. There was a stepwise increase in recurrence rate (RR) among patients with increasing SPF category (RR: 20%, 41%, and 53%). Patients whose SPF categories varied, from low or intermediate in one part of the tumor to high in another, seemed to have a poor prognosis (RR = 57%).(ABSTRACT TRUNCATED AT 250 WORDS)