How ferritin-Fe becomes available for cell functions is unknown. Our previous studies with rat hepatoma cells indicated ferritin had to be degraded to release its Fe. Here we investigated whether this occurs in other cell types and requires lysosomes. Release of ferritin-Fe was induced with desferoxamine (DFO) in ⁵⁹Fe-preloaded hepatoma, Caco2, and erythroid K562 cells and measured by rocket immunoelectrophoresis and autoradiography. Half-lives for ferritin-⁵⁹Fe and protein were parallel (23, 16, and 11h for the hepatic, Caco2 and K562 cells, respectively). Co-treatment with 180 µM Fe, leupeptin, chymostatin or chloroquine markedly decreased rates of ferritin-Fe release and ferritin degradation. Lactacystin had no effect except a small one in erythroid cells. Fractionation of hepatoma cell lysates on iodixanol gradients showed rapid depletion of cytosolic ferritin by DFO treatment but no accumulation in lysosomes. We conclude that regardless of cell type, release of Fe from ferritin occurs mainly through lysosomal proteolysis.