Flow cytometric multi-parameter analysis has proven to be a powerful tool to characterize subpopulations of cell suspensions, and is applied routinely in hematology. However, in studies of cancer where there is interest in defining phenotypic markers in conjunction with DNA content, this method has hardly been applied [6]. Our objective was to develop a methodology that extends previous investigations on relative and absolute quantitation of the epidermal growth factor receptor (EGFR) in dual parameter analysis in vitro on human bladder cancer cell lines [3]. In order to quantitate EGFR content in tumours and to relate it to DNA content, tumour selection, DNA-content, and EGFR-content measurements should be carried out simultaneously. Different fluorescent dyes were used to optimize DNA assessment and antibody staining, using a single laser instrument as a practical approach for clinical routine. In vitro cultures were used to validate the quality of tumour cell selection and antigen quantitation. Therefore, two urothelial tumour cell lines-lowly and highly differentiated-were incubated under different conditions: monolayer (ML), three-dimensional multi-cellular spheroids (MCS) and cocultures (COCU) with the fibroblast cell line NI were investigated and EGFR quantitation was related to S-phase fraction (SPF). Accurate determination of instrument settings allows simultaneous three-colour analysis with DNA assessment. Tumor cell selection based on staining with phycoerythrine (R-PE) against a highly expressed urothelial glycoprotein, detected with the antibody Uro5 or against cytokeratin appeared to be possible in FL2, using the fluorochrome combination fluorescein-isothiocyanate (FITC), R-PE and propidiumiodide (PI). Using this staining protocol, relative and absolute EGFR quantitation (quantum simply cellular beads) is shown to be accurate, when FITC is used for EGFR staining and measured in the green fluorescence channel (FL1). Using this colour combination EGFR content and SPF of tumour cells were compared in different growth states, and could be monitored reliably. In spite of a higher emission spectrum of 7-aminoactinomycin-D (7-AAD), this DNA stain provided no advantage over PI. Broad coefficients of variation (CV) were found when intact cells were stained, thus hindering accurate assessment of ploidy and S-phase fraction. Similarly, Syto-13, a DNA dye detected in FL1, could not be optimized for multi-parameter measurements. Although the emission maximum is at 520 nm, the spectrum is too wide to compensate fluorescence overlap in FL2 or FL3. Quantum Red (QR), used as a streptavidin conjugate in FL3, could not be combined with two other colours for DNA staining, since sufficient compensation was not obtainable when an argon ion laser is used. The coculture model allows verification of tumour cell selection and discrimination. The high differentiated tumour cell line RT4 shows an unambiguous correlation between EGFR content and S-phase fraction. The low differentiated tumour cell line J82 presents a similar pattern of post-transcriptional EGFR regulation with respect to culture condition, however, the S-phase fraction is basically unaffected.