Proteins are required for the efflux of heme from mitochondria and liposomes. The efflux from liposomes is independent of the heme-binding affinity of the protein (Biochem. 23: 3715, 1984). We tested whether heme-binding proteins increase efflux of newly synthesized heme from structurally and functionally intact rat liver mitochondria. Mitochondria whose heme was labeled with ¹⁴C-δ-aminolevulinic acid, were incubated in the presence of glutathione transferases (GSTs), serum albumin (RSA) or heme-binding protein (HBP), all from the rat. HBP caused a 6–8 fold increase in efflux of newly synthesized heme as compared to that effected by RSA or GSTs. This result indicates that heme efflux from intact mitochondria, unlike that from liposomes, depends on the type of protein present and that HBP may specifically facilitate heme efflux from mitochondria.