Circulating extracellular microvesicles (cEVs) are characterised by presenting surface antigens of parental cells. Since their biogenesis involves the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane, exposed PS has been considered as a recognition hallmark of cEVs. However, not all cEVs externalise PS. In this study, we have phenotypically and quantitatively characterised cEVs by flow cytometry, paying special attention to the proportions of PS in chronic heart failure patients (cHF; n = 119) and a reference non-HF group (n = 21). PS--cEVs were predominantly found in both groups. Parental markers showed differential pattern depending on the PS exposure. Endothelium-derived and connexin 43-rich cEVs were mainly PS--cEVs and significantly increased in cHF. On the contrary, platelet-derived cEVs were mostly PS+ and were increased in the non-HF group. We observed similar levels of PS+- and PS--cEVs in non-HF subjects when analysing immune cell-derived Evs, but there was a subset-specific difference in cHF patients. Indeed, those cEVs carrying CD45+, CD29+, CD11b+, and CD15+ were mainly PS+-cEVs, while those carrying CD14+, CD3+, and CD56+ were mainly PS--cEVs. In conclusion, endothelial and red blood cells are stressed in cHF patients, as detected by a high shedding of cEVs. Despite PS+-cEVs and PS--cEVs representing two distinct cEV populations, their release and potential function as both biomarkers and shuttles for cell communication seem unrelated to their PS content.Keywords: annexin V; connexin 43; endothelial cells; extracellular vesicles; heart failure; leukocytes; microvesicles; phosphatidylserine; platelets; red blood cells.