In erythroid cells iron uptake from transferrin (Tf) is utilized largely for heme synthesis. Here we provide evidence that Tf receptor (TfR) expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine erythroleukemia (MEL) cells with 5-aminolevulinic acid (ALA) resulted in an increase in TfR expression accompanied by enhanced uptake of iron from Tf and incorporation of iron into heme. ALA-mediated enhancement of TfR mRNA expression was completely prevented by succinylacetone, an inhibitor of ALA dehydratase, and N-methylprotoporphyrin, an inhibitor of ferrochelatase, indicating that the effect of ALA required its metabolism to heme. Treatment of cells with ALA was associated with enhanced iron regulatory protein-2 (IRP-2) binding activity, which could be blocked by inhibitors of heme synthesis and supplementation of the culture medium with a permeable iron chelate or Tf. In all cases, IRP-2 activities were correlated exactly with TfR mRNA levels. Thus, in addition to the previously characterized transcriptional up-regulation of TfR expression in differentiating erythroid cells, increased TfR expression mediated by enhanced heme biosynthesis may ensure sufficient iron availability for optimal heme synthesis and prevent possible protoporphyrin accumulation under conditions of inadequate iron supply.