We sought to establish the relationship of quantitative hepatic iron measurements and phlebotomy-mobilized iron in a large sample of HFE C282Y homozygotes with a hemochromatosis phenotype. Thus, we analyzed data from 79 unrelated C282Y homozygotes from treatment centers in Rochester, NY and Birmingham, AL who had undergone liver biopsy with measurement of hepatic iron content and who had achieved iron depletion (serum ferritin <25 ng/l) with quantitative phlebotomy. The sample consisted of 57 men and 22 women; their median age at diagnosis was 47 years (range 23-76 years). Sixty-three of 79 (79.7%) had hepatic iron index (HII; μmol/g dry weight of liver divided by age in years) ≥1.9, a conventional phenotypic definition of hemochromatosis. The mean quantity of phlebotomy-mobilized iron (± 1 sd) was 6.4 g (±4.0 g) in men (range 2.0-18.0 g) and 6.2 g (±5.8) in women (range 0.7-22.5 g). There was a significant positive correlation of liver iron levels with phlebotomy-mobilized iron in this patient sample (Pearson coefficient 0.75; R<PRE>2</PRE>=55.5%). This relationship was also demonstrable when data from males and females were analyzed separately. We calculated a phlebotomy-mobilized iron index (MII: phlebotomy-mobilized iron in mg divided by age in years) using the corresponding regression equations and evaluated its use as a surrogate for HII. Thus, a phlebotomy-mobilized iron of 3.5 g corresponds to liver iron levels of 80 μmol/g dry weight, and a MII of 80 corresponds to HII of 1.9. Forty-six of 79 subjects met all four phenotypic criteria for hemochromatosis (liver iron levels ≥80 μmol/g, HII≥1.9, phlebotomy-mobilized iron ≥3.5 g and MII≥80). Of the 20 subjects with MII<80, 9 had a HII≥1.9. Conversely, 5 of 16 subjects with HII<1.9 had MII≥80 and 8 had phlebotomy-mobilized iron ≥3.5 g. Most patients with a hemochromatosis phenotype and evidence of moderate or severe iron overload (>80%) are homozygous for the common HFE missense mutation C282Y. Thus, clinicians rely increasingly on HFE mutation analysis to diagnose hemochromatosis and on quantitative phlebotomy to estimate the severity of iron overload in many cases. Liver biopsy is now employed in selected patients to visualize fibrosis or cirrhosis and to identify coincidental hepatic disease. We conclude that the use of the MII permits a retrospective estimation of the age-adjusted severity of iron overload that has a diagnostic value similar to that of the HII in hemochromatosis patients with C282Y homozygosity.