Although most cellular iron is firmly bound (e.g. in haemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The LIP is regarded as the crossroads of cellular iron traffic. Using multi-parameter flow-cytometry of cells treated with the metal-sensitive sensor calcein and the cell-permeable chelator deferiprone, we studied LIP in various human erythroid cell populations in peripheral blood, bone marrow and culture. Erythroid maturation was found to be associated with a decrease in the LIP. In the peripheral blood, nucleated erythrocytes (normoblasts) had 5.8-fold and 8.8-fold greater LIP than reticulocytes and erythrocytes respectively. Early reticulocytes had 2.5-fold more LIP than late reticulocytes. In the bone-marrow and in culture, LIP decreased by c. 30-fold as early erythroid precursors matured to late precursors. Adding holo-transferrin to iron-depleted cultures elevated LIP by 3.9-fold. We also show that in beta-thalassaemia, a disease associated with iron-overload, erythrocytes and reticulocytes in the blood and erythroid precursors in culture have a significantly greater LIP than their normal counterparts. In conclusion, the LIP in erythroid cells is altered under physiological (maturation) and pathological (thalassaemia) conditions. The methodology presented might be useful for evaluating the LIP in various diseases and for studying the efficacy of iron-chelators.