BACKGROUND The percentage of hypochromic red blood cells (RBCs) (%HYPO) has been demonstrated as the best predictor of response to iron loading in haemodialysis patients treated with recombinant human erythropoietin (rHuEPO). However, we have previously shown that this parameter is positively influenced by erythropoietic activity since reticulocytes are considered hypochromic by cell counters. New cell counters are able to determine cell volume and haemoglobin (Hb) concentration separately on reticulocytes and mature erythrocytes. The aim of this study was to assess the sensitivity and specificity of mature erythrocyte parameters in detecting functional iron deficiency (FID). METHODS A total of 32 stable chronic haemodialysis patients in the maintenance phase of rHuEPO therapy were included. Classical parameters of iron monitoring and mature erythrocyte parameters were measured after a 4-week iron-free period. Patients were classified as responders (R) or non-responders (NR) to an iron load of 100 mg iron sucrose at each dialysis session for 4 weeks, according to whether their Hb increased by >1 g/dl at the end of iron loading. RESULTS Twelve patients were identified as responders. Receiver operating characteristic (ROC) curve analysis demonstrated %HYPO and its corresponding parameter on mature erythrocyte, %HYPOm, as the best predictors of FID. The other parameters were ordered as follows: tranferrin saturation (TSAT), ferritin (FRT), mature RBC Hb content (CHm), mean corpuscular Hb concentration (MCHC), percentage of mature erythrocytes with a low CHm (%lowCHm), mean content in Hb (MCH) and reticulocyte Hb content CHr. Comparing the parameters at different cut-offs, the best sensitivity, specificity and efficiency were demonstrated for %HYPOm> 6%. CONCLUSIONS The best efficiency to predict FID was found for %HYPOm> 6%. The predictive value of %HYPO was quite similar. The clinical impact of %HYPOm in iron monitoring should also be tested in the induction phase of rHuEPO treatment because of its independence from erythropoietic activity.