Prerequisites for successful flow cytometry investigations are specific antibodies labeled with appropriate fluorochromes and negligible autofluorescence of the untreated cells at the wavelength of interest. The aim of this study was (a) to characterize frequently used urological carcinoma cell lines with regard to their autofluorescence properties, (b) to demonstrate the autofluorescence as a serious interfering factor on FACS analysis of urological carcinoma cell lines and (c) to suggest an alternative to avoid interfering autofluorescence. Twenty-one cell lines originating from prostate carcinoma, renal cell carcinoma and bladder cancer were included in this study. The various cell lines were read on a flow cytometer in comparison to human erythrocytes as cells with low fluorescence intensity. Urological cell lines show a high autofluorescence when flow cytometry analyses are performed at the frequently used excitation wavelengths at 405 and 488 nm. At excitation wavelength of 633 nm, this problem was reduced and most of the cell lines (14/21) were without autofluorescence at the emission wavelength of 785 nm. In addition, with a spectrofluorometer three exemplary cell lysates were investigated. The above observations were confirmed. The dye APC-Cy7 is one suitable fluorochrome for successful investigation under these measurement conditions.