K562 cells were used to investigate the factors that control hemoglobin (Hb) synthesis. Treatment with sodium butyrate enhanced Hb synthesis and glycophorin A expression. delta-Aminolevulinate synthase (ALAS) activity and Hb levels simultaneously increased to a similar extent and with a similar time course, and the increases were dependent on the concentration of diferric transferrin (FeTf) in the culture medium. Addition of exogenous delta-aminolevulinic acid (ALA) resulted in a dose-dependent increase in Hb content. Hb synthesis was inhibited 50% after addition of succinylacetone (SA), a potent inhibitor of delta-aminolevulinate dehydratase. These findings suggest that ALAS is a key enzyme in the eight steps of de novo heme synthesis and that iron, including FeTf, plays a central role in Hb synthesis through control of ALAS activity in erythroid differentiating cells. On the other hand, erythropoietin (EPO) treatment had no effect on Hb synthesis and slightly suppressed glycophorin A expression. Hemin enhanced Hb synthesis in the K562 cells but not glycophorin A expression. The addition of ALA, SA, or FeTf to hemin-treated cells caused no significant changes in Hb synthesis. Butyrate, EPO, and hemin acted on the K562 cells in different ways and caused different biochemical changes in the Hb synthesis process.