To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [⁵⁵Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [⁵⁵Fe]hemin binding sites, concomitant with the increase in [³H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP eDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a K_d of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [⁵⁵Fe] hemin, the binding to PBR had a K_d of 15±1.8 nM. The B_max of [⁵⁵Fe] hemin binding to the mitochondria was 6.88±0.76 pmol/mg of protein, a value consistent with that of [³H] PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.