The purpose of these experiments was to determine whether chronic dietary iron overload causes impairment of hepatic mitochondrial and/or microsomal calcium sequestration. Experimental iron overload was produced by feeding three groups of rats a chow diet supplemented with 3.0% (wt/wt) carbonyl iron for up to 8 weeks achieving graded increases in hepatic iron concentrations ranging from 1360 to 3170 micrograms/g. At low levels of iron overload, there were no changes in mitochondrial oxidative metabolism or calcium sequestration, whereas at moderate and high degrees of iron loading, both of these parameters were significantly reduced. In contrast, there were significant decreases in microsomal cytochrome P450 levels and microsomal calcium sequestration at all three levels of iron loading. These abnormalities occurred at hepatic iron concentrations at which the authors have previously found evidence of hepatic organelle lipid peroxidation. These alterations in organelle calcium sequestration may impair intracellular calcium homeostasis in the liver and contribute to subsequent cellular injury.