L1210 leukemic cells can be cultured continuously in serum-free medium supplemented merely with either transferrin or iron salts. No transferrin or transferrin-like molecules were detected in the conditioned medium from cells established in serum-free medium plus iron. In these cells, iron uptake was found to occur through a saturable transport system exhibiting the properties of an allosteric regulatory protein. This transferrin-independent iron transport coexisted with transferrin-mediated iron uptake. When the iron concentration in the medium is less than 0.1 microM, transferrin must be present in the culture medium in order to observe cell growth. Under these culture conditions, a 16- to 18-h treatment with a 1 mM concentration of the iron chelator desferrioxamine resulted in less than 20% DNA synthesis compared to control cultures. DNA synthesis was reinitiated without a lag time after addition of 1 mM ferric citrate to the culture medium. No heme synthesis was needed to observe this DNA synthesis. However, in the presence of the antioxidant propyl gallate the reinitiation of DNA synthesis was abolished. Ferricyanide could not replace ferric citrate as a stimulant of DNA synthesis. Cytofluorometric analysis has shown that nearly 10% of the cells treated by desferrioxamine were blocked in G2 + M phase of cell cycle, suggesting that, in addition to DNA synthesis, iron chelation also blocked other mechanisms critical for cell growth.