AbstractCirculating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CV_L), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CV_L of CD34bright on CEC ~ 30%; CV_L of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (median_female = 9.31 CEC/mL; median_male = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.