Flow cytometric (FCM) DNA analysis yields information on ploidy status and the S‐phase fraction (SPF), variables of prognostic importance in breast cancer. The clinical value of the SPF is currently being evaluated in prospective randomized trials. The widespread use of FCM DNA analysis emphasizes the importance of reproducibility (both intra‐ and interlaboratory). In this study, 67 nuclear suspensions of breast cancer samples were analyzed by 12 laboratories routinely performing FCM DNA analysis in breast cancer. No general guidelines were imposed; each laboratory used its own standard protocols. For DNA ploidy status (diploid vs. non‐diploid), agreement was complete for 79% (53/67) of the samples, compared with 64% (43/67) of samples when tetraploidy was considered [i.e., euploid (diploid + tetraploid) vs. aneuploid (the remaining non‐diploid)]. For the SPF, pairwise comparison of the results of all 12 laboratories yielded a mean Spearman's rank correlation of 0.78 (range: 0.54–0.93). For those 39 samples being categorized in low or high SPF by all laboratories, all agreed in 14 samples (36%). Similar patterns were obtained with kappa measures, agreement being good for ploidy status (diploid vs. non‐diploid; overall K = 0.87 and 0.74 for euploid vs. aneuploid), but moderate for the SPF [overall k = 0.47 (for low SPF vs. high SPF vs. “no SPF reported”)]. Discrepancies were chiefly attributable to differences in the categorization of the S‐phase values, rather than in FCM procedures, other critical differences being in the detection and interpretation of near‐diploid and small non‐diploid cell populations, the definition of tetraploidy, and the choice and execution of the method used for S‐phase estimation. Based on the observations of this study, detailed guidelines for FCM analysis and interpretation of data are proposed in the Appendix. Some issues remain, however, e.g., to standardize a method for S‐phase calculation and tetraploid definition.© 1995 Wiley‐Liss, Inc.