A conserved motif, termed the heme regulatory motif (HRM), was identified in the presequences of the erythroid δ-aminolevulinate synthase precursors and was shown to be involved in hemin inhibition of transport of these proteins into mouse mitochondria in vitro. When the HRM was inserted into the presequence of the ornithine transcarbamoylase precursor, a normally unregulated mitochondrial protein, it conferred hemin inhibition on the transport of the chimeric protein. The conserved cysteine within the HRM was shown by site-directed mutagenesis to be required for hemin inhibition.