Hemochromatosis has often been associated with progressive iron overload, but the natural history of iron accumulation in untreated C282Y homozygotes has been reported infrequently. The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 101 168 primary care participants for iron overload using transferrin saturation, unbound iron-binding capacity, Serum ferritin (SF), and HFE C282Y and H63D genotyping. SF was measured at initial screening (IS) and again when selected participants returned for a clinical examination (CE). The change in SF over the observation period (defined as ferritin rate of change) was analyzed according to age, gender, initial SF, initial SF/age, transferrin saturation, and iron removed by phlebotomy in C282Y homozygotes. Seventy-four male and 133 female untreated C282Y homozygotes were observed over a median of 112 days (34-924 days) between IS and CE. In men, SF increased in 54% and decreased in 46%. In women, SF increased in 50% and decreased in 50%. The significant variables affecting the SF rate were initial log SF (P = 0.0027) and transferrin saturation (P < 0.0001). Male C282Y homozygotes with higher SF rates (n = 27, upper 50th percentile) had significantly greater iron removed by phlebotomy (mean 4.93 g, range 1.0-17 g) than those with lower SF rates (n = 26, lower 50th percentile) (mean 2.6 g, 0.42-7.1, P < 0.05). SF was as likely to decrease as increase in untreated C282Y homozygotes over this relatively brief observation period. Incremental increases in SF are not inevitable in untreated C282Y homozygotes.