We initiated these studies to help clarify the roles of heme, ␦-aminolevulinic acid (ALA), hemA, and hemM in Escherichia coli heme synthesis. Using recombinant human hemoglobin (rHb1.1) as a tool for increasing E. coli's heme requirements, we demonstrated that heme is a feedback inhibitor of heme synthesis. Cooverexpression of rHb1.1 and the hemA-encoded glutamyl-tRNA (GTR) reductase increased intracellular levels of ALA and heme and increased the rate of rHb1.1 formation. These results support the conclusion that heme synthesis is limited by ALA (