The assessment of iron status for hemodialysis patients has been hindered by the inaccuracy of commonly used diagnostic tests. A novel assay, the reticulocyte hemoglobin content (CHr), has recently been found to sensitively detect functional iron deficiency among nonuremic patients treated with recombinant erythropoietin (rHuEPO). The purpose of this study was to evaluate the CHr for the assessment of iron status in hemodialysis patients. One hundred sixty-four stable hemodialysis patients had a mean CHr of 27.5 +/- 2.8 pg with a normal distribution of values. The mean CH (mature red cell hemoglobin content) was 26.4 +/- 2.4 pg. There was a close correlation between CHr and CH (r = 0.86, P < 0.0001). A significant subgroup of patients (12.2%) had CHr values < CH. These patients had recent increases in rHuEPO dose, and a lower mean transferrin saturation and hematocrit, suggesting the recent onset of functional iron deficiency due to the increase in rHuEPO dose. In the second phase of the study, 32 patients were randomly selected to receive treatment with a single dose infusion of 1,000 mg of intravenous iron dextran (IVFe). Patients were classified as iron deficient (N = 7) if they responded with a significant reticulocytosis (sustained 1 basis point increase in corrected reticulocyte index within 2 weeks). All other patients were classified as iron replete (N = 25). A CHr < 26 pg at baseline predicted iron deficiency with a sensitivity of 100%, specificity of 80%. The serum ferritin, transferrin saturation and percentage of hypochromic red blood cells all were less accurate. The time to correction of iron deficiency at the level of the reticulocyte was found to be within 48 hours as measured by correction of the mean CHr to > 26 pg, and by the shift of the vast majority of the reticulocyte population to CHr > 26 pg within this time span. We conclude that CHr < 26 pg is an accurate measure of iron status in hemodialysis patients, that a CHr value < CH indicates the acute onset of iron deficiency, and that a single dose infusion of intravenous iron results in correction of iron deficiency at the level of the reticulocyte within 48 hours.