Cyclins are important in the regulatory system controlling cell cycle progression and overexpression or aberrant expression of G1 cyclins, as cyclin E, may result in an inadequate G1-S transition. In this study, the expression of cyclin E was analyzed in 12 human cell lines and 18 hematological malignancies by flow cytometry, and protein levels were quantified using calibrating microbeads. The majority of the tested cell lines demonstrated a cell cycle-specific expression of cyclin E, even though some cell lines expressed lower and more constant cyclin E levels throughout the cell cycle. No correlation between the fraction of cyclin E-positive cells and cyclin E content could be observed in the cell lines. Similar cyclin E levels were observed in the tumor samples as for the cell lines, although the fractions of positive cells were low. The flow cytometric quantification of cyclin E was compared with densitometric quantification of cyclin E Western blots, and a significant correlation between the two methods was observed (rs = 0.56, P = 0.03). The variation of cyclin E levels between cell lines was significantly higher than the variation for repetitive analyses of the same cell line, suggesting that the expression of cyclin E could reliably be quantified using flow cytometry. Because of the potential to analyze specific subgroups such as cyclin E-positive cells, this method may facilitate the detection of cyclin E-overexpressing tumor cells.