BACKGROUND: We previously developed a real-time quantitative RT-PCR technique to detect breast carcinoma cells in peripheral blood (PB). The aim of the current study was to improve cytokeratin 19 (CK19) quantification using plasmid dilutions of cloned PCR fragments to obtain a more reliable and reproducible quantification of CK19 transcripts. MATERIALS AND METHODS: PB samples of 14 stage IV breast cancer patients and 23 healthy controls were examined with RT-PCR using plasmid quantification. RESULTS: Median CK19+ copy numbers of one and 11 were detected in the control group and stage IV breast cancer patients, respectively (Mann-Whitney, P </=0.0001). When comparing the results obtained using cell line dilutions with those obtained using plasmid dilutions, a good correlation was observed (r(2) = 0.98). CONCLUSIONS: Plasmid dilutions are more reliable than cell line dilutions for quantification of gene expression, and more objective criteria for positivity could be defined based on the characteristics of the standard curve (slope and intercept). A more universally accepted agreement on the definition of the cut-off value for positivity is needed.